It was evaluated physical, antibacterial and antibiofilm properties of Zataria multiflora Boiss essential oil (ZEO) and its nanoemulsion. Long-term stability of nanoemulsion prepared by emulsion phase inversion was satisfying based on low narrow size distribution (polydispersity index 0.2) and low droplet size (200nm) over 21 days of storage. Nanoemulsion showed lower minimum inhibitory concentration (MIC) on Listeria monocytogenes (2500 mu gmL(-1)) than Salmonella Typhimurium (5000 mu gmL(-1)). Killing kinetics study revealed that nanoemulsion was more effective in inhibiting the growth of bacteria in milk than culture media. Both bacteriostatic and bactericidal effects were observed depending on the type of bacteria, nanoemulsion concentration and the time of exposure. Nanoemulsion at 4xMIC concentration reduced 64% and 75% of one-day-old biofilm of L.monocytogenes and S.Typhimurium, respectively. In conclusion, nanoemulsion revealed antimicrobial activity, but converting the ZEO to nanoemulsion did not improve its antibacterial activity; however, antibiofilm properties were enhanced.