Abstract(#br)In this study, organic acids were used as cross-linker with polyvinyl alcohol (PVA) films prepared by a solution-casting method for food packing applications. The effect of incorporating three different organic acids, i.e., malic acid (MA), tartaric acid (TA), and lactic acid (LA), on the physicochemical and biological properties of PVA was explored in detail. The crystalline phase, optical absorption, and functional groups were examined via UV–Vis and Fourier transform infrared spectroscopy. Thermal, microstructural, and surface investigations were conducted by thermogravimetric analysis and scanning electron microscopy, and the antibacterial activity was evaluated. The surface topography and roughness were found to have a strong effect on the bactericidal properties of the films, as confirmed by atomic force microscopy. Among the considered films, PVA/LA exhibited the highest bacterial inhibition, which was largely due to its capacity to modify the local pH and alter the permeability of the microbial layer by disrupting bacteria–substrate interaction. In general, the composite film was found to have attractive properties and can be considered as a food packaging material with low environmental impact based on polyvinyl alcohol.