Photocatalytic activity in titanium dioxide (TiO₂) has been extensively studied because of its potential use in sterilization, sanitation, and remediation applications. The aim of the study reported here was to assess the feasibility of “fixed” TiO₂ as the photocatalyst for inactivating pathogenic bacteria selected, Staphylococcus aureus and Escherichia coli, from a water stream. The investigation was undergone in a properly designed laboratory-scale evaluation. Using the system reported here, we obtained an effective bactericidal capability for E. coli and S. aureus with 90.0% and 98.0% after 30 and 10 min ultraviolet-A light irradiation with fixed TiO₂, respectively. Parameters such as the various initial bacteria concentration, TiO₂ concentrations, interruption of illumination, turbidity, and coexisted organic matters were examined to identify the removal efficiency in the photocatalytic reaction. Results indicated the negative effect by high bacteria concentration, coexisted organic matters, and turbidity on inactivation of bacteria, and positive effect on disinfection was associated with higher TiO₂ concentration. Furthermore, our results indicated that under the same experimental conditions, the removal efficiency of the system in synthetic water was performed better than that of crude water. This inferior removal capability in crude water is mainly caused by the negative effect from the unknown coexisted factors.