Herein, we constructed a nano-CuO film on copper foil for the in situ generation of reactive chlorine species (RCS) for the contact killing of bacteria and biofilm suppression and dispersal. Both Staphylococcus aureus and Escherichia coli were susceptible to this treatment. Additionally, we demonstrate the solid nano-CuO interface-based heterogeneous Fenton chemistry rather than leached copper-ion-based homogeneous Fenton chemistry is responsible for RCS generation and bacterial cytotoxicity. This process is modulated by the H2O2 and Cl- concentrations in bacterial growth environment. The potential applications for in situ generation of RCS might be the incorporation of CuO nanoparticles into functional food contact surfaces as well as coating materials for marine biofouling control.