[Objective] This study aimed to study the effect of different free chlorine concentration and exposure time on bacterial inactivation as well as cellular adenosine triphosphate (ATP). [Methods] Inactivation behaviors of autochthonous bacteria from river water were investigated with flow cytometry (FCM). Total cell counts (TCC), intact cell counts (ICC) and intracellular and extracellular (ATP) were analyzed during the chlorination process. [Results] The results showed that both chlorine concentration and exposure time influenced the removal efficiency of bacteria. At a low concentration of chlorine (<2 mg/L), a prolonged exposure time was needed to achieve a high removal efficiency of bacteria. In contrast, at a high concentration of chlorine (≥2 mg/L), inactivation of more than 90% of bacteria could be achieved within a short exposure time (i.e. 1 min). The results also indicated high nucleic acid (HNA) content bacteria were more vulnerable than low nucleic acid (LNA) content bacteria during the chlorination process. Furthermore, it was observed that concentration of intracellular ATP decreased with increasing chlorine doses. However, the increase of extracellular ATP was only recorded at high chlorine doses (≥2 mg/L). [Conclusion] Chlorination resulted in a decrease of ICC, and reduction of bacterial activity with increasing chlorine concentration. The results demonstrated that FCM and ATP measurements are useful and fast tools to assess inactivation kinetics of indigenous riverine bacteria during the chlorination treatment.