The packaging system is one of the most challenging researches, in which antibacterial agents were embedded. In this research activity, we used folic acid (FA), as a biosafe and biodegradable substance for the surface modification of zinc oxide nanoparticles (ZnO NPs). Then, the ZnO-FA NPs as nanofillers were inserted in the poly(vinyl alcohol) (PVA) with different weight percentages (2, 5, and 8 wt%, relative to polymer weight). The synthesized substances were characterized using various analyses like Ultraviolet-Visible (UV-Vis) spectroscopy, water contact angle, field emission scanning electron microscopy, and thermogravimetric analysis. The image of transmission electron microscopy analysis displayed the excellent dispersal of the ZnO-FA NPs within the polymeric matrix. By increasing the percentage of the modified ZnO NPs into the PVA, the intensity of the absorption peaks in the UV-Vis spectra was raised, and the thermal stability of NC films got better compared to the pure polymer. PVA/ZnO-FA NC film 8 wt% showed better antibacterial activity than the pure PVA and had the best resistance against Grampositive bacteria (Staphylococcus aureus).