A new composite antibacterial material ZnO/Cu2+-Chitosan/Montmorillonite (ZCCM) was prepared with montmorillonite as carrier, Zn(Ac)2•2H2O, Cu(NO3)2•3H2O and chitosan as raw materials. ZCCM was characterized by X-ray diffraction, nitrogen physical adsorption, scanning electron microscopy and energy dispersion spectrometry. The antibacterial activity of ZCCM against Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus was evaluated by minimal inhibitory concentration, minimum bactericidal concentration and the influence of growth curves. ZCCM displays excellent antibacterial activity which is higher than ZnO-Montmorillonite, Cu2+-Montmorillonite and ZnO/Cu2+-Montmorillonite. In addition, the antibacterial mechanism of ZCCM was investigated by analyzing bacterial morphology, integrity of cell membrane, lipid peroxidation and the effect of histidine on antibacterial activity of materials. It is found that cell morphologies of bacteria are damaged and bacterial cells are shrunken. With the increase of cell membrane permeability, the intracellular dissolved matters leak continuously. What's more, the reactive oxygen species are generated and biomacromolecules are oxidized. [ABSTRACT FROM AUTHOR]