This study deals with preparation and evaluation of properties of chitosan/zinc oxide bionanocomposites (CT/ZnO BNCs) with different amounts of modified zinc oxide nanoparticles (ZnO NPs) through ultrasonic irradiation technique. Due to the high surface energy and tendency for agglomeration, the surface ZnO NPs was modified by a coupling agent as 3-aminopropyltriethoxysilane (APS) to form APS-ZnO nanoparticles. Fourier transform infrared (FTIR) spectroscopy confirmed that APS was successfully grafted onto the ZnO nanoparticles surface. Thermogravimetric analysis (TGA) revealed a surface coverage of the coupling molecule of 2.6wt%. The resulting bionanocomposites were characterized by FTIR spectra, X-ray diffraction patterns, and TGA. The antibacterial activity of bionanocomposite films was tested against gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The results of CT/ZnO BNCs revealed that the thermal and antibacterial properties obviously improved the presence of ZnO NPs in comparison with the pure CT and that this increase is higher when the NP content increases. Further, it was observed that antibacterial activity of the resulting hybrid biofilms showed somewhat higher for gram-positive bacteria compared to gram-negative bacteria.