In this investigation, the effect of different concentrations of titanium dioxide (TiO2) nanoparticles (NPs) on the structure and antimicrobial activity of chitosan-based coating films was examined. Analysis using scanning electron microscopy (SEM) and atomic force microscopy (AFM) revealed that the modified TiO2 NPs were successfully dispersed into the chitosan matrix, and that the roughness of the chitosan-TiO2 nanocomposites were significantly reduced. Moreover, X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analyses indicated that the chitosan interacted with TiO2 NPs and possessed good compatibility, while a thermogravimetric analysis (TGA) of the thermal properties showed that the chitosan-TiO2 nanocomposites with 0.05% TiO2 NPs concentration had the best thermal stability. The chitosan-TiO2 nanocomposite exhibited an inhibitory effect on the growth of Escherichia coli and Staphylococcus aureus. This antimicrobial activity of the chitosan-TiO2 nanocomposites had an inhibition zone ranging from 9.86 ± 0.90 to 13.55 ± 0.35 (mm). These results, therefore, indicate that chitosan-based coating films incorporated with TiO2 NPs might become a potential packaging system for prolonging the shelf-life of fruits and vegetables.