Unravelling the antibacterial properties and mechanisms of action of essential oil nanoemulsions: A literature review

1.1.1 Inclusion criteria and study selection

Studies were considered for inclusion if they were original research articles or reviews published in peer-reviewed journals. The master focus was on literature that investigated the formulation of EOs into NEs and subsequently evaluated their antibacterial activity, with a specific interest in those exploring the underlying MsOA. No restrictions were placed on the specific essential oil studied to capture the breadth of the field, but the focus remained on studies involving pathogenic bacteria.

1.1.2 Exclusion criteria

Research that exclusively examined antifungal or antiviral activity, studies on non-bacterial pathogens, studies published in languages other than English, and studies lacking mechanistic information were excluded. This methodology was employed to provide a structured and transparent framework for the review, allowing readers to understand the basis of the literature discussed while acknowledging the narrative synthesis of the findings presented.

3.1.1 Structural classification

NEs are nanoscale dispersions of two unblendable liquids, either oil-in-water (o/w) or water-in-oil (w/o). Unlike coarse emulsions, which appear milky-white, they are typically transparent or translucent. NEs are heterogeneous dispersions of two unmixable or immiscible liquids, stabilized by surfactants, with droplet sizes generally ranging from 20 to 200 nm. These systems are particularly advantageous for EO delivery due to their enhanced stability, improved water dispersibility, and increased bioavailability resulting from the large surface-to-volume ratio of nanometric droplets. The three primary constituents of NEs are oil or lipid phase, water or aqueous phase, and surfactant. The creation and stability of these emulsions rely on both the physicochemical characteristics of these components and their correct formulation (Barradas et al., 2021; Preeti et al., 2023).

A W/O NE is characterized by water droplets dispersed within a continuous oil phase, while an O/W NE features oil droplets within a continuous aqueous phase. Emulsifier selection is critical; lipophilic emulsifiers coat droplets in W/O systems, and hydrophilic emulsifiers are employed for O/W systems. The O/W type is well-suited for creating edible coatings, as it enables the incorporation of various lipophilic chemicals or compounds with antimicrobial and antioxidant properties into a hydrophilic polymeric network (Fig. 1). The common fabrication method involves homogenizing a high-melting lipid with a hydrophilic surfactant in water at an elevated temperature, next by cooling to induce crystallization of the oil droplets (Mushtaq et al., 2023).

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Fig. 1. Nanoemulsions components and forms.

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The selection of suitable ingredients is crucial for the effective formulation of NEs. The practice uses Generally Recognized As Safe (GRAS) components, including surfactants (ex, tweens, spans) and biopolymers like food proteins and polysaccharides, owing to their minimal harm. Edible films and coatings are generally classified according to their primary structural constituent, which may be lipids, proteins, polysaccharides, or a composite. To improve chemical stability, three main approaches are employed: the addition of antioxidants or chelating agents, the manipulation of the interface (its charge, reactivity, and thickness), and the control of atmospheric conditions for example temperature, pH, oxygen, and light (Liu et al., 2019; Sridhar et al., 2021).

NEs are broadly classified into two primary types based on the nature of the stabilizing agent employed at the oil-water interface. Conventional Nes are thermodynamically metastable systems stabilized by molecular surfactants, which lower interfacial tension and form a protective layer to retard droplet coalescence; these surfactants can be synthetic, such as the widely used Tween (non-ionic) and Span series, or biosurfactants like rhamnolipids and surfactin, which offer an eco-friendly and biocompatible alternative. In contrast, Pickering Emulsions (PEs) are stabilized by solid nanoparticles or nanocomplexes (e.g., silica, chitosan, or protein-polysaccharide complexes) that adsorb irreversibly at the interface, creating a robust physical barrier against coalescence and Ostwald ripening due to their high desorption energy. The choice of stabilization system including synthetic surfactants, biosurfactants, or solid particles is critical, as it dictates not only the kinetic stability and rheology of the NE but also its application suitability in fields ranging from food and pharmaceuticals to cosmetics, with Pickering systems often providing superior long-term stability and reduced need for chemical surfactants (Gauthier and Capron 2021; Kumar and Mehta 2025; El-Sakhawy et al., 2025b).

3.2.1 Techniques for nanoemulsion formulation

The preparation of NEs involves two primary categories: (low and high, LE & HE)-energy methods. HE techniques utilize processes like high-pressure homogenization, microfluidization, and ultrasonication. In contrast, LE formulation methods, such as phase inversion emulsification and self-nanoemulsification, are typically preferred for their greater efficiency and lower instrumental complexity, as they consume less energy. However, for food-grade emulsions, HE methods are often more suitable because they necessitate smaller amounts of surfactant. Furthermore, the formulation techniques for NE drug delivery systems, especially low-energy ones, frequently exhibit overlapping characteristics (Kumar et al., 2019).

6.1.1. NE formulation increases the surface area

EONE formulation increases the surface area or makes nano droplets that come into proximity with the CM, which facilitates cellular Interaction (Fig. 2).

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Fig. 2. Key mechanisms and impact of the formulation enhancement of EOs as nanoemulsions against bacteria.

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The formulation of essential oil into a NE increases the surface area by creating nanodroplets that come into close proximity with the CM, thereby facilitating cellular interaction. Reducing the essential oil into nanoscale droplets (typically <200 nm) dramatically increases the total surface area available for interaction with bacterial cells (da Silva et al., 2022). Due to their nanometric dimensions of NE, the droplets have a heightened surface area, thereby improving the likelihood of chemical interactions between bioactive compounds and targets such as bacterial cells or free radicals (da Silva et al., 2023c). Compared to traditional formulations like conventional emulsions, lipid NEs containing crude oil from medicinal plants or hydrophobic drugs have been demonstrated to improve drug solubility, decrease side effects of various potent drugs, increase the bioavailability of drugs, and prolong the pharmacological properties (Uhumwangho and Okor, 2005).

The antimicrobial NE is thought to work by using its tiny lipid particles to deliver the EO directly to the E. coli cellular membrane (CM). This proximity allows the oil’s hydrophobic components to disrupt the membrane’s integrity, potentially by damaging its phospholipid bilayer or hindering its transport proteins. Consequently, the membrane becomes leaky, releasing vital intracellular contents like nucleic acids, proteins, and potassium, leading to cell death within five minutes. The NE is significantly more effective than pure essential oil because its small, suspended particles can easily reach the membrane, unlike the pure oil, which has poor water solubility and cannot interact with the cells as efficiently (Moghimi et al., 2016). Therefore, the smaller EO NE droplet size correlates with higher antibacterial efficiency. In addition, the specific size range of the NE droplets may enhance its versatility, allowing it to act as an effective inhibitor against bacteria of various sizes. This broadens its spectrum of antibacterial activity and increases its overall bioactivity.

6.1.2. Nanodroplets enter the cells through porin channels or passive absorption

The NEs were characterized by droplet sizes of < 200 nm, making them at least ten times smaller than vegetative bacterial cells. This small size allows them to enter cells via porin channels or passive absorption. Once inside, the release of NE contents through mechanisms like mass transport, adhesion, and diffusion disrupts the CMs of bacteria, causing bacterial cellular death (Tayeb et al., 2022). Porin proteins, which are abundant hydrophilic transmembrane channels in Gram-negative bacteria, permit small NE droplets with hydrophilic surfaces to pass via the CM (Nazzaro et al., 2013; How et al., 2024).

The antibacterial activity of NE is boosted by two simultaneous mechanisms. First, their improved solubility increases the concentration gradient of active compounds around bacterial cells. Second, the nanodroplets are small enough to passively enter the cells through porin channels. Together, these actions make NEs more effective (da Silva et al., 2023a).

6.1.3. Improved solubility and delivery

NEs have appeared promising delivery vehicles for EOs. EOs are inherently hydrophobic, and NE technology encapsulates the oil in surfactant-stabilized droplets, effectively dispersing it in water and enabling better delivery of the active compounds to bacterial targets. This improved delivery system means that lower concentrations of the EO are required to achieve the same or even greater antibacterial effect compared to the free oil (Kumar et al., 2025b).

NEs are kinetically stable colloidal systems with tiny droplet sizes, granting them improved functional properties over conventional emulsions. This advantage has spurred interest in applying them to develop lipophilic substances utilizing diverse fatty acids (Al-Sabagh et al., 2011; Begum et al., 2024).

6.1.4. Nanoemulsion formulation enhanced disruption of cell membrane integrity

NEs can enhance the disruption of membranes, a process linked to the similarity in the chemical nature (lipophilic or hydrophilic) between the NE components and the CM lipids. The key principle at play is the “like dissolves like” adage, which facilitates interaction and destabilization (Ali et al., 2025). 

The small, HE droplets in a NE can fuse with and destabilize the lipid bilayers of bacterial membranes. This disruption compromises membrane integrity, leading to leakage of critical intracellular components for instance potassium ions, proteins, and nucleic acids, ultimately causing cell death. This mechanism is often passive and nonspecific, making it difficult for bacteria to develop resistance (Hamouda and Baker, 2000; Sugumar et al., 2013).

One possible explanation for the increased antibacterial activity of citral o/w NEs against P. aeruginosa is that the outer lipopolysaccharide membrane engages in electrostatic interactions with the aqueous phase of the NE, thereby enabling easier passage of the hydrophobic oil components. Support for this mechanism comes from the observation that NEs stabilized with Lantana camara, a source of hydrophilic compounds, were particularly effective at killing P. aeruginosa. Conversely, the lipophilic ends of lipoteichoic acid on the surface of Gram-positive S. aureus limit NE penetration because of unfavorable interactions (Hyldgaard et al., 2012; Guliani et al., 2021).

NE droplets are engineered to merge with microbial membranes through electrostatic attraction between the cationic NE and the typically anionic bacterial surface, as well as thermodynamic processes. This fusion allows the droplets to integrate into the phospholipid bilayer. Once a critical number of droplets have fused, their stored energy and the release of active agents disrupt the membrane’s integrity by forming pores, leading to the uncontrolled leakage of cellular contents and making this mechanism particularly effective against bacteria (Donsì et al., 2012; Begum et al., 2024).

In addition to these two potential mechanisms, the antibacterial effectiveness increases as the NE droplet size decreases. Furthermore, the specific size range of the droplets enhances their versatility, enabling them to act as effective inhibitors against a broad spectrum of bacteria of various sizes. Formulating EOs as NEs transforms them into more potent antibacterial agents not by altering their chemical composition, but by enhancing their physical properties. The nanometric size improves delivery and interaction with bacteria, leading to more efficient and rapid disruption of the microbial CM.

6.2.1 Fusion and electrostatic interaction

NE droplets are designed to fuse with the lipid microbial membranes. This fusion is driven by electrostatic attraction between the typically negatively charged bacterial surface and cationic components of the NE, as well as by thermodynamic mechanisms. Once a sufficient number of nanoparticles fuse with the pathogen, the energy trapped within the NE, combined with the released active components, compromises the lipid membrane’s integrity (Begum et al., 2024). This process is particularly effective against bacteria because the droplets can integrate into the phospholipid bilayer, creating pores and causing uncontrolled leakage of intracellular bacterial contents (Donsì et al., 2012).

6.2.2 Consequences of membrane disruption

The fusion event and subsequent membrane disruption have several catastrophic consequences for the bacterial cell:

(1) Increased permeability

The integrity of the CM is lost, leading to a rapid leakage/passage of vital ions (such as K⁺, Na⁺, Ca²⁺), ATP, nucleic acids, and proteins. Exposure to the NE caused substantial rupture of the bacterial CMs, resulting in a swift leakage of bacterial cell components (Hemmila et al., 2010; Duan et al., 2022; Alvand et al., 2023). The minute size of NE particles results in an extensive surface area and lowered surface tension. These characteristics enable increased interaction with cells and take advantage of the heightened permeability of biological membranes, leading to improved bioavailability (Baspinar and Borchert 2012). This EONE leakage depletes the cell of essential molecules and energy, halting metabolism. The medication accumulated less in organs like the liver and spleen when it was incorporated into NEs containing unesterified ethyl oleate, lecithin, and modified egg yolk because the mononuclear phagocyte system absorbed the drug less readily (Brussel et al., 2012).

(2) Loss of cytosolic content

Generally, severe rupture of the membrane results in the efflux of larger cytoplasmic materials, ultimately leading to cell lysis and death (Iranpour et al., 2025). Eugenol NEs, as an example of EONEs, have demonstrated severe deformation and membrane rupture in Escherichia coli and Staphylococcus aureus as visualized by scanning electron microscopy (Fu et al., 2022).

(3) Inhibition of biofilm formation

Biofilms are not just a cluster of bacteria; they are a protected, organized community embedded in a self-produced matrix. This structure creates physical and chemical barriers that shield the embedded bacteria from antibiotics, allowing the biofilm to survive at concentrations that would easily kill individual, planktonic cells. By targeting the CM, NEs can prevent bacterial adhesion and biofilm formation, a key virulence factor for many bacterial pathogens. They have also shown efficacy in removing already adhered cells, making them potent antibiofilm agents (Raj et al., 2022).

6.3.1 Oxidative stress (OS) and lipid peroxidation (LP)

Oxidative stress and resulting lipid peroxidation play a critical role in the antibacterial activity of both the host immune system and many antimicrobials, ultimately leading to bacterial cell death. The accumulation of reactive oxygen species (ROS) damages bacterial cell components, with polyunsaturated fatty acids in CMs being a primary target for peroxidation (Vaishampayan et al., 2021).

A significant biochemical mechanism involves the induction of OS. Treatment with certain NEs, such as those containing eugenol or polymethoxy-flavones, triggers a sharp increase in intracellular levels of reactive oxygen species (ROS) in bacterial cells. In another study, through oxidative stress and bacterial membrane damage, cinnamon EONE may demonstrate antibacterial properties (Zhao et al., 2023; Chen et al., 2025). Generally, OS, induced by the excessive production and build-up of ROS, leads to the impairment of cellular components and their functions. An overaccumulation of ROS leads to oxidative damage of cellular components, most notably the peroxidation of membrane lipids. This lipid peroxidation degrades the phospholipid bilayer, further increasing membrane fluidity and permeability, and culminating in the breakdown of the membrane structure (Demidchik 2015; Su et al., 2019). The measurement of elevated malondialdehyde (MDA), a key biomarker for lipid peroxidation, confirms this mechanism in bacteria treated with eugenol NEs (Fu et al., 2022).

6.3.2 Interference with cellular metabolism

NEs can interfere with important bacterial metabolic pathways, according to advanced metabolomic investigations. For instance, it was discovered that a polymethoxyflavones-eugenol NE significantly alters the metabolomic profile of E. coli, thereby halting bacterial development (Chen et al., 2025).

6.3.3 EONEs-mediated inhibition of gene expression or nucleic acid damage

EONEs can cause the leakage or damage of intracellular nucleic acids (DNA and RNA) as part of their antimicrobial MOA. The NE format enhances this effect compared to the bulk essential oil due to better cellular penetration. 

Shiga toxins (Stx1a and Stx2a) are critical bacterial virulence factors, notable for their extreme potency and their MOA, which involves the inhibition of host cell protein synthesis. Inhibition of shiga toxins (Stx1a and Stx2a) gene expression, indicating in nucleic acid production (Melton-Celsa, 2014). Research demonstrates that a NE form of Zataria multiflora Boiss EO exhibits superior anti-E. coli activity compared to its pure essential oil counterpart. Gene expression analysis revealed that at 75% MIC, the NE down-regulated the transcription of stx1A and stx2A by 4.75 and 4.80-fold after 72 hours, a significantly stronger suppression than that caused by the pure oil. The enhanced efficacy of the NE in curtailing both bacterial proliferation and Shiga toxin production (Azizkhani et al., 2020).

According to Abd-Rabou and Edris (2022), frankincense essential oil from Boswellia sacra, which is high in α-pinene, can induce apoptosis in A549 lung cancer cells and may help prevent recurrence. The oil’s anticancer effect was significantly increased when formulated into a water-based NE using propylene glycol as a co-surfactant. This formulation was highly toxic to cancer cells but had minimal impact on healthy cells, suggesting its potential as a plant-based adjuvant for lung cancer chemotherapy (Abd-Rabou and Edris, 2022). The study also implies that certain EONEs could target bacterial DNA.

In the same context, an in vitro investigation demonstrated that the essential oil of Nigella sativa induces cell death and apoptosis in liver cancer cells. When formulated into a NE, its anti-cancer efficacy was enhanced, as shown by reduced cell proliferation, a lower IC50, increased apoptosis, and higher expression of pro-apoptotic genes. The surfactant type in the NE was a key factor in boosting this cytotoxicity. Both the crude EO and its NEs were non-toxic to normal cells, indicating selective toxicity against cancer cells (Abd-Rabou and Edris, 2021).

The study by Ghani et al. (2019) found that while E. coli exhibited resistance to pure cinnamon essential oil, while a NE of the oil demonstrated significantly greater antibacterial efficacy. This NE, formulated with soy protein and lecithin to create droplets of 141.2 nm, caused substantial damage to the CMs of both E. coli and E. faecium. The mechanism was evaluated by measuring the leakage of intracellular components, including nucleic acids, proteins, and potassium ions. Treatment with the NE at the Minimum Inhibitory Concentration (MIC) resulted in a marked increase in the release of these cellular materials compared to both the control and pure oil treatments. This was evidenced by an approximate two-fold increase in the optical density of the bacterial culture filtrates. The findings indicate that the NE ‘s enhanced effectiveness is due to greater compromise of membrane integrity, leading to the loss of large molecules, including nucleic acids, and subsequent bacterial death (Ghani et al., 2019).

6.3.4 Disrupting efflux function and inhibition of other vital processes

Time-kill assays of lemon essential oil NEs (NEs-LEO) revealed a significant reduction in bacterial viability after 5 hours of exposure at 1x MIC (6.25 mg/mL). According to flow cytometry, LEO-NEs disrupted the bacterial CM potential, compromised its integrity, and impaired efflux pump activity. Further investigation using confocal laser scanning microscopy showed that a higher concentration (8x MIC) altered E. coli CM permeability and cell wall integrity. Proteomic analysis proposed that the antibacterial mechanism involves the enhancement of bacterial chemotaxis and a marked inhibition of ribosomal assembly. Additional metabolic pathways potentially impacted include those for butyric acid, ascorbic acid, aldehyde metabolism, and the sulphur-relay system. In summary, LEO-NEs exhibit a strong bacteriostatic effect on E. coli, primarily by permeabilizing and depolarizing the CM, disrupting efflux function, and targeting ribosomal assembly and chemotaxis pathways (Xiao et al., 2024).

6.3.5 Inhibition of virulence factors

The microevolution of drug-tolerant bacteria, driven by antibiotic overuse, stands as a major challenge of this century. As a survival strategy, bacteria often form biofilms, which significantly increase their resistance to antimicrobial drugs. Bacterial biofilms in wounds can block drug delivery and delay healing. A recently identified approach to managing these difficult infections involves preventing biofilm formation and inhibiting the virulence factors of multidrug-resistant pathogens (Mirghani et al., 2022).

Researchers developed a hydrogel containing an Eucalyptus EONE. This NE, physically cross-linked with Carbomer 940 and carboxymethyl chitosan, demonstrated effective antibacterial and anti-biofilm action against S. aureus in lab tests. The resulting hydrogel dressing exhibited ideal properties for wound care, including good rheology, water retention, and biocompatibility. In animal studies, the treatment successfully accelerated wound healing, reduced bacterial counts, and promoted the regeneration of skin tissue, showing significant promise for managing S. aureus-infected wounds (Cai et al., 2023).

Stable EONEs and their compounds offer a promising solution for inhibiting virulence and biofilm formation in planktonic bacteria like Pseud. aeruginosa and Acine. baumannii. These NEs show superior anti-biofilm and anti-virulence effects compared to commercial antibiotics by suppressing the expression of genes essential for pathogenicity, biofilm development, and surface attachment. When incorporated into hydrogel coatings, these NEs exhibit powerful antibiofilm properties, making them suitable for application on healthcare equipment such as catheters, ventilators, and hospital beds to prevent biofilm-related infections (Raj et al., 2022).

According to Balasubramanian et al. (2023), Listeria monocytogenes, a significant foodborne pathogen in the United States, forms resilient biofilms on various surfaces under different temperatures. Their research demonstrates that a eugenol NE is effective at both preventing biofilm formation and eradicating established biofilms on stainless steel at 10°C and 25°C. The MOA involves the suppression of key virulence traits, including bacterial motility, the secretion of extracellular polymeric substances, extracellular DNA (eDNA) abundance, and quorum-sensing activity. This anti-biofilm effect was consistent across two tested strains (Scott A and AT19115). The study concludes that eugenol NE has potential as a natural disinfectant in food industry settings, and future work correlating biofilm reduction with decreased food contamination would further validate its efficacy (Balasubramanian et al., 2023).

Using an environmentally friendly process, researchers successfully formulated both a clove oil-emulsion and a clove oil-NE. The NE consisted of spherical nanoparticles and demonstrated superior antimicrobial properties compared to the conventional emulsion. It exhibited a lower MIC against pathogenic Gram-negative and S. aureus Gram-positive bacteria. Furthermore, the clove oil-NE was significantly more effective at inhibiting S. aureus biofilm formation and showed stronger antifungal activity against various strains, including Candida albicans and Aspergillus species (Shehabeldine et al., 2023).

Shiga toxins (Stx1a and Stx2a) are highly potent bacterial virulence factors that cause severe disease by inhibiting protein synthesis in host cells (Melton-Celsa, 2014). A study found that a NE of Zataria multiflora Boiss EO was more effective at inhibiting E. coli growth than the pure essential oil. After 72 hours, a 75% MIC dose of the NE reduced the expression of the stx1A and stx2A genes by 4.75-fold and 4.80-fold, respectively, which was a greater reduction than that achieved by the pure oil. These results indicate that the NE has significant potential as a “green” food-grade preservative, due to its ability to suppress both bacterial growth and the production of key virulence factors (Azizkhani et al., 2020).

6.3.6 Synergistic effects of EONEs with antibiotics

According to Wei et al. (2023), a NE of tea tree EO acted synergistically or partially synergistically with antibiotics to combat multidrug-resistant bacteria, both Gram-(positive and negative). In cells infected with drug-resistant E. coli, these combinations also strengthened the intestinal barrier by improving electrical resistance and increasing the production of tight junction proteins. In vivo experiments demonstrated that combining the NE with amoxicillin enhanced weight gain and preserved the structure of the intestinal wall. The MOA was linked to a reduction in bacterial adhesion and invasion, as the NE suppressed key proteins and genes (fimC, fimG, fliC) responsible for E. coli’s fimbriae and flagella, ultimately damaging the bacterial CM.

The EOs from T. vulgaris, L. maroccana, and A. leucotrichus showed promising antibacterial properties. Using a mixture design approach, an optimal blend was identified, consisting of 55% T. vulgaris, 41% L. maroccana, and 4% A. leucotrichus. This specific combination was highly effective, demonstrating low minimum inhibitory concentrations (MICs) against E. coli, Staph. aureus, and Pseud. aeruginosa. Furthermore, the optimal mixture acted synergistically with antibiotics, reducing the MIC of gentamicin by four times and that of amoxicillin by a factor of 2 to 64. When formulated as a NE, this blend was more potent than any of the individual EOs. These findings indicate that this optimized EO combination, especially in a NE or paired with traditional antibiotics, could be a valuable strategy for combating certain drug-resistant bacteria (Soulaimani et al., 2022).

The NEs of Citronella and Mentha spicata EOs exhibited antibacterial effects by targeting the cell surface (E. coli) and disrupting the bacterial membrane. Electron microscopy revealed that exposure to the oils, particularly at a 2x MIC concentration, caused significant cell deformation and damage, with higher concentrations producing more pronounced effects (Ragab et al., 2022).