Microbial contamination of most foods occurs primarily at the surface during post-processing and handling; therefore, preventing cross-contamination by incorporation of antimicrobial substances in contact with the surface of the product is an efficient strategy in reducing food contamination risks. Zinc Oxide (ZnO) particles have been used widely to achieve antibacterial films in various applications including in the food industry. This work describes the fabrication of antimicrobial multilayered polymeric films containing Zinc Oxide nanoparticles by the co-extrusion technique. Effects of various film thicknesses (1.5-15 µm) containing Zinc Oxide nanoparticles on the antimicrobial effectiveness of the film by their capability to inactivate DH5α Escherichia coli were studied. The results show that developed films have the potential to be used as food packaging films that can extend shelf life, maintain quality, and assure the safety of food. The antibacterial mechanisms of ZnO nanoparticles were also investigated and it was found that direct contact of the particles with products is necessary to assure high antibacterial activity of films.