Packaging is very important to maintain the quality of food and prevent the growth of microbes. Therefore, the use of food packaging with antimicrobial properties protects the food from the growth of microorganisms. In this study, antibacterial nanocomposite films of polyvinyl alcohol/starch/chitosan (PVA/ST/CS) together with nickel oxide-copper oxide nanoparticles (NiO-CuONPs) are prepared for food packaging. NiO-CuONPs were synthesized by the co-precipitation method, and structural characterization of nanoparticles (NPs) was carried out by XRD, FTIR, and SEM techniques. Composites of PVA/ST/CS, containing different percentages of NPs, were prepared by casting and characterized by FTIR and FESEM. The mechanical properties, diffusion barrier, and thermal stability were determined. The nanoparticles have a round structure with an average size of 6.7 ± 1.2 nm. The cross-section of PVA/ST/CS film is dense, uniform, and without cracks. In the mechanical tests, the addition of NPs up to 1% improved the mechanical properties (TS = 31.94 MPa), while 2% of NPs lowered TS to 14.76 MPa. The fibroblast cells toxicity and the films antibacterial activity were also examined. The films displayed stronger antibacterial effects against Gram-positive bacteria (Staphylococcus aureus) compared to Gram-negative bacteria (Escherichia coli). Furthermore, these films have no toxicity to fibroblast cells and the survival rate of these cells in contact with the films is more than 84%. Therefore, this film is recommended for food packaging due to its excellent mechanical and barrier properties, good antibacterial activity, and non-toxicity.