BACKGROUND There is a growing demand in the food industry for the replacement of synthetic preservatives with their natural alternatives. This has led to the development of novel methods such as encapsulation of plants essential oil with appropriate physicochemical stability, and antibacterial and organoleptic properties. This study aimed to prepare an optimal nanoemulsion of Thymus daenensis L. essential oil for use as a natural preservative in mayonnaise. RESULTS The analysis of droplet diameter, polydispersity index, zeta potential, encapsulation rate, and intrinsic stability showed that out of nine T. daenensis essential oil‐containing nanoemulsions, two preparations of A and B had high stability scores. In vitro antibacterial tests showed the adverse effect of Tween 80 volume on the antibacterial properties of nanoemulsions. One nanoemulsion (essential oil:Tween 80, ratio 1:1, 15 min sonication) was considered to be optimal based on its long‐term stability and antibacterial effects on Salmonella Typhimurium, Escherichia coli, and Listeria monocytogenes. However, compared to the optimal nanoemulsion, the pure essential oil showed more antibacterial effects. The bacterial control in mayonnaise was close to equal for sodium benzoate (in maximum limit 1 g kg−1) and the optimal nanoemulsion (½ MIC) for 24 h. The optimal nanoemulsion achieved significantly higher sensory scores (taste, appearance, and mouthfeel) than the pure essential oil in mayonnaise (P < 0.05). CONCLUSION The results demonstrated similar antibacterial effects for the optimal nanoemulsion and sodium benzoate. The optimal nanoemulsion, due to its desirable sensorial attributes, long‐term stability, and slow release of volatile compounds, can be considered an appropriate alternative to synthetic preservatives.