The inactivation kinetics of E. coli and MS2 coliphage by singlet oxygen (¹O₂) were investigated in a homogeneous photosensitization system using Rose Bengal (RB) and visible light illumination (the Vis/RB system). The inactivation of E. coli and MS2 in the Vis/RB system was monitored over time with variations of several parameters such as pH, light intensity, concentration of RB, and the presence of dissolved oxygen. In addition, the concentration of ¹O₂ generated by the Vis/RB system was quantified using furfuryl alcohol under each microbial inactivation conditions. Based on the obtained results, the degree of microbial inactivation was quantitatively correlated with ¹O₂ exposure using the (delayed) Chick-Watson model. The Ct (concentration-time product) values of 1O2 required for 2 log microbial inactivation were found to be 1.3×10−4 mg·min/L for E. coli and 1.9×10⁻⁵ mg·min/L for MS2, respectively. The inactivation of E. coli exhibited an initial lag phase until 0.5×10−4 mg·min/L of Ct.