In this paper, a novel S-scheme CuS/Bi5O7I heterojunction was successfully constructed using a two-step approach comprising the alkaline hydrothermal method and the adsorption–deposition method, and it consisted of Bi5O7I microrods with CuS particles covering the surface. The photocatalytic antibacterial effects on Escherichia coli (E. coli) were systematically examined with visible light exposure. The results suggested that the 3%-CuS/Bi5O7I composite showed the optimal antibacterial activity, completely inactivating E. coli (5 × 108 cfu/mL) in 180 min of irradiation. Moreover, the bacterial inactivation process was scientifically described. •O2− and h+ were the major active species for the inactivation of the bacteria. In the early stages, SOD and CAT initiated the protection system to avoid the oxidative destruction of the active species. Unfortunately, the antioxidant protection system was overwhelmed thereafter, which led to the destruction of the cell membrane, as evidenced by the microstructure changes in E. coli cells. Subsequently, the leakage of intracellular components including K+, proteins, and DNA resulted in the unavoidable death of E. coli. Due to the construction of the S-scheme heterojunction, the CuS/Bi5O7I composite displayed the boosted visible light harvesting, the high-efficiency separation of photogenerated electrons and holes, and a great redox capacity, contributing to an outstanding photocatalytic disinfection performance. This work offers a new opportunity for S-scheme Bi5O7I-based heterojunctions with potential application in water disinfection.