Improving the antibacterial activity of biodegradable materials is crucial for combatting widespread drug-resistant bacteria and plastic pollutants. In this work, we studied polyaniline (PANI)-functionalized zinc oxide nanoparticles (ZnO NPs) to improve surface charges. A PANI-functionalized ZnO NP surface was prepared using a simple impregnation technique. The PANI functionalization of ZnO successfully increased the positive surface charge of the ZnO NPs. In addition, PANI-functionalized ZnO improved mechanical properties and thermal stability. Besides those properties, the water permeability of the bionanocomposite films was decreased due to their increased hydrophobicity. PANI-functionalized ZnO NPs were applied to thermoplastic starch (TPS) films for physical properties and antibacterial studies using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The PANI-functionalized ZnO bionanocomposite films exhibited excellent antibacterial activity for both E. coli (76%) and S. aureus (72%). This result suggests that PANI-functionalized ZnO NPs can improve the antibacterial activity of TPS-based bionanocomposite films.