The cupric ion mediated inactivation of Escherichia coli was enhanced by the presence of hydrogen peroxide (H2O2), with increasing inactivation efficacy observed in response to increasing concentrations of H2O2. The biocidal activity of the Cu(II)/H2O2 system is believed to result from the oxidative stress caused by reactive oxidants such as the hydroxyl radical (•OH), cupryl species (Cu(III)), and the superoxide radical (O2•–), which are produced via the catalytic decomposition of H2O2. In E. coli cells treated with Cu(II) and H2O2, the intracellular level of •OH and Cu(III) increased significantly, leading to complete disruption of cell membranes. On the basis of experimental observations made using an •OH scavenger, copper-chelating agents, and superoxide dismutase, it is concluded that Cu(III) is the predominant species responsible for the death of E. coli cells. It was also found that the production of Cu(III) was promoted by the reactions of copper with intracellular O2•–. MS2 coliphage was found to be even more susceptible than E. coli to the oxidative stress induced by the Cu(II)/H2O2 system.