In this study, antimicrobial activity of chitosan and whey protein isolate edible films incorporated with different concentrations of zataria multiflora Boiss essential oil; ZEO (1%, 2%, 3% and 4% v/v) and zataria multiflora Boiss essential oil nanoliposome (0, 1, 2, 3 and 4%) against Listeria monocytogenes (ATCC 19118), Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 65138), Escherichia coli O157: H7 (ATCC 35218) and vibrio parahaemolyticus (ATCC 43996) were evaluated using disc-diffusion test. The circular disks containing ZEO and NZEO were cut and placed on bacterial lawn. Plates were incubated for 24 h at 35o C. The inhibition zone diameter was measured by digital caliper. These inhibition zones were formed against Listeria monocytogenes (ATCC 19118), Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 65138), Escherichia coli O157: H7 (ATCC 35218) and vibrio parahaemolyticus (ATCC 43996). The results of this study indicated that by increasing the concentration of ZEO in both whey protein isolate and chitosan films, a significant increase was observed in inhibition zones’ diameters (P<0.05). The greatest antimicrobial effect was achieved at 4 % concentration of essential oil against Listeria monocytogenes in both films. Whereas films containing NZEO did not show any significant antimicrobial activities at the highest concentrations. According to the results of the GC/MS analysis, Zataria multiflora Boiss. essential oil is a rich source of different antimicrobial compounds especially thymol and carvacrol which might be considered as the main reasons of the observed antimicrobial activities in the formulated biofilms. The results of this study indicated that whey protein isolate and chitosan edible films containing Zataria multiflora Boiss essential oil showed a good antimicrobial effect and this may be considered as an advantage to use them as an active packaging ingredient in food products.