The antibacterial activity of ZnO nanoparticles (nano ZnO) on various microorganisms has been investigated in this study and acting mechanism is proposed for E. coli a model organism by analyzing the growth, permeability and morphology of the bacterial cells. The experimental results indicated 1 mmoL (81.4 μg) nano ZnO could completely inhibit the growth of 10 7 cfu/mL bacterial cells in liquid Luria-Betrani medium. In the biochemical study, it has been observed that, nano ZnO resulted in the leakage of proteins. Chemiluminescence assay proved the generation of the reactive oxygen species into active state. When the cells of E. coli were exposed to nano ZnO, many pits were observed in bacterial cells by TEM and Fluorescence microscopy and the cells were found to be fragmentary. Released zinc ion from has been monitored by AAS. It suggested that nano ZnO is able to destroy the permeability of the bacterial membranes. Our study demonstrated that nano ZnO damages the structure of bacterial cell membrane and depressed the activity of some membranous enzymes by ROS production which caused E. coli bacteria to die eventually. Copyright © 2011 American Scientific Publishers All rights reserved.