•Reverse micelles in vegetable oils were used as nanocarriers to encapsulate nisin.•Structural system characterization revealed some changes in the presence of nisin.•The loaded systems showed antimicrobial efficacy against pathogens in real foods.•The nanocarriers’ antimicrobial efficacy depended on food matrices’ composition.•Ethanol may play an important role on the interactions with food matrices. Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers’ constituents (probably ethanol). Our findings suggest complex interactions that take place when RMs are applied in different food matrices.