We propose a green approach for the size-controlled synthesis of Cu₂O nanoparticles (NPs) with simplified chemical deposition technology by adjusting the concentration of the precursor copper source. The morphologies, phase compositions and structures were recorded for the obtained Cu₂O NPs with sizes of 10, 50, 100 and 200 nm. In the dark, all of the Cu₂O NPs showed good antibacterial activity towards Escherichia coli K-12 (E. coli). A size-dependent antibacterial effect was also observed as a result where decreasing the size of the Cu₂O NPs led to increasing the antibacterial activity. The smallest Cu₂O NPs (Cu₂O-10) with the minimum inhibitory concentration (≤1 µg mL⁻¹) achieved the highest antibacterial efficiency, with the inactivation of 7-log E. coli in 80 min with a concentration of 5 µg mL⁻¹ in the dark. Under visible light (VL), medium concentrations (10, 20 and 50 µg mL⁻¹) of Cu₂O-10 showed enhanced antibacterial activity compared to that in the dark. For low (5 µg mL⁻¹) or high concentrations (200 µg mL⁻¹) of Cu₂O-10, the antibacterial activity under VL was almost the same as that in the dark. The mechanisms of antibacterial activity are proposed and discussed in detail. “Contact killing” and photogenerated h⁺ together with reactive oxygen species (such as ˙OH and H₂O₂) were found to be responsible for bacterial inactivation in the dark and under irradiation, respectively. Cu₂O-10 proved to be a good photocatalyst, capable of the highly efficient evolution of H₂O₂, whose stable equilibrium concentration could be as high as 150 µM (using 20 µg mL⁻¹ of Cu₂O-10). Study on the membrane-separated system shows that the concentration of diffusing H₂O₂ can be as high as 50 µM, contributing to a complete inactivation of 7-log E. coli in 7 h. For comparison, the inactivation kinetics of the Cu₂O NPs and H₂O₂ were calculated, and could be fitted into the Weibull and shoulder-linear-tail models, respectively.