Zinc oxide (ZnO) nanoparticles (NPs) exhibit antibacterial activity against both Gram-positive and Gram-negative bacteria. However, the antimicrobial mechanism of ZnO NPs remains unclear. In this study, we investigated the interactions among ZnO NPs, released chemicals (Zn2 + and Reactive Oxygen Species, ROS) and Escherichia coli (E. coli) cells. ZnO NPs without contacting with bacterial cells showed strong antibacterial effect. The results of the leakage of intracellular K+ and integrity of carboxyfluoresce in-filled liposomes showed that ZnO NPs have antimicrobial activity against E. coli by non-specifically disrupting E. coli membranes. Traces of zinc ions (1.25 mg/L) and hydrogen peroxide (from 1.25 to 4.5 μM/L) were detected in ZnO NPs suspensions, but was insufficient to cause the antibacterial effect. However, the addition of radical scavengers suppressed the bactericidal effect of ZnO coated films against E. coli, potentially implicating ROS generation, especially hydroxyl radicals, in the antibacterial ability of ZnO NPs.