The photocatalytic bactericidal activity of titanium dioxide (TiO2) thin films has been extensively studied. In this study, we investigated the bactericidal activities of TiO2 nanotube (NT) thin films using Escherichia coli and Staphylococcus aureus cells as the model bacteria. Metallic titanium (Ti) thin films were anodized on a silicon (Si) wafer substrate to form TiO2 NT thin films. To evaluate the bactericidal activity of the TiO2 NT thin films, bacteria on the TiO2 NT thin films were irradiated with near-ultraviolet light (UV-A) at a wavelength of 365 nm. The bactericidal activity was estimated by the survival rate derived from the number of live cells, which form colonies on the cell culture medium. We demonstrated that the survival rate of the two types of bacteria investigated in this study was significantly reduced by UV light irradiation and that there was a difference in the temporal change in the survival rate between the two types of bacteria. Furthermore, we investigated the generation of reactive oxygen species (ROSs) by UV light irradiation of TiO2 NT thin films using electron spin resonance spectroscopy and fluorescence analysis. We found that the main ROS generated on the surface of the TiO2 NT film was the hydroxyl radical, OH•. In addition, the generation of ROSs increased with an increase in the UV irradiation time. We proposed a kinetic model that reproduces the dependence of bacterial viability on the UV light irradiation time by considering the temporal change in the amount of ROSs generated by UV light irradiation. A comparison of the calculated and experimental results revealed that the bactericidal effect consisted of the direct photolysis of bacteria and the photocatalysis via the generation of hydroxyl radicals, with the latter exhibiting a stronger bactericidal effect than the former.