•Microemulsions based on four essential oils were used to encapsulate nisin.•Structural changes were revealed regarding the micellar interface flexibility.•Nisin loaded systems showed antimicrobial efficacy against pathogens.•Dittany-based microemulsions had the best antimicrobial efficacy against pathogens.•The system’s antimicrobial efficacy depended on microemulsions’ characteristics. During the last years, the food industry is working on the replacement of high energy methodologies with more sustainable techniques for the encapsulation of natural preservatives, in order to enhance their effectiveness as food additives. In the present study, nisin, an antimicrobial agent, was encapsulated in essential oil-containing microemulsions. More specifically, rosemary, thyme, oregano, and dittany essential oil-containing microemulsions were formulated to encapsulate nisin enhancing the system’s overall antimicrobial activity. The systems were investigated for the interfacial properties and size of the surfactants’ monolayer using electron paramagnetic resonance spectroscopy and dynamic light scattering. Subsequently, nisin-loaded microemulsions were tested for their antimicrobial activity against Lactococcus lactis, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus, using the well diffusion assay. Finally, this technique was validated by a killing assay. Overall, this study provides important information on the antibacterial activity of nisin-loaded nano-carriers enhanced by essential oils, in relation to the microemulsions’ structure.