A series of PLA/ZnO bionanocomposite films were prepared by introducing positively surface charged zinc oxide nanoparticles (ZnO NPs) into biodegradable poly(lactic acid) (PLA) by the solvent casting method, and their physical properties and antibacterial activities were evaluated. The physical properties and antibacterial efficiencies of the bionanocomposite films were strongly dependent on the ZnO NPs content. The bionanocomposite films with over 3% ZnO NPs exhibited a rough surface, poor dispersion, hard agglomerates, and voids, leading to a reduction in the crystallinity and morphological defects. With the increasing ZnO NPs content, the thermal stability and barrier properties of the PLA/ZnO bionanocomposite films were decreased while their hydrophobicity increased. The bionanocomposite films showed appreciable antimicrobial activity against and . Especially, the films with over 3% of ZnO NPs exhibited a complete growth inhibition of E. coli. The strong interactions between the positively charged surface ZnO NPs and negatively charged surface of the bacterial membrane led to the production of reactive oxygen species (ROS) and eventually bacterial cell death. Consequently, these PLA/ZnO bionanocomposite films can potentially be used as a food packaging material with excellent UV protective and antibacterial properties.