Under dark conditions, we systematically study on antibacterial mechanism of ZnO nanoparticles and found that the antimicrobial activity of ZnO nanoparticles results from the attachment of ZnO to bacterial cell walls and subsequent release of Zn2+ ions to the bacterial cytoplasm. Display omitted To evaluate the antibacterial activity of zinc oxide (ZnO) in the absence of a light source, we examined three different types of nanoparticles that vary in size and the number of oxygen defect sites. Colony forming units (CFU) and various microscopic investigations revealed that the antibacterial activity of ZnO nanoparticles under dark conditions was not related to reactive oxygen species (ROS) generation and nanoparticles transfection, but depended on the ZnO attachment to bacterial cell walls and increasing concentrations of Zn2+ ions in the bacterial cytoplasm due to local dissolution of the attached ZnO.