Chlorination and UV illumination are being widely applied to inactivate a number of pathogenic microbials in the environment. Here, we evaluated the inactivation efficiency of individual and combined treatments of chlorination and UV under various aqueous conditions. UV dosage was required higher in waste water than in phosphate buffer to achieve the similar disinfecting efficiency. Free chlorine generated by electrolysis of waste water was abundant enough to inactivate microbials. Based on these, hybrid system composed of sequential treatment of electrolysis-mediated chlorination and UV treatment was developed under waste water conditions. Compared to individual treatments, hybrid system inactivated bacteria (i.e., E. coli and S. typhimurium) and viruses (i.e., MS-2 bacteriophage, rotavirus, and norovirus) more efficiently. The hybrid system also mitigated the photo re-pair of UV-driven DNA damages of target bacteria. The combined results suggested the hybrid system would achieve high inactivation efficiency and safety on various pathogenic microbials in wastewater.