Silver-based antibacterial agents have obtained wide attention due to the fact that bacteria in the environment is ubiquitous, which has become one of the most difficult problems for human health. However, the antibacterial mechanism and process are still inconclusive. Here, Ag2O nanoparticles (NPs) with uniform spherical morphology and small size (around 30 nm) were prepared. The as-prepared Ag2O NPs induced high antibacterial activity (100% inhibition ratio) against E. coli. A two-step antibacterial process was proposed and confirmed, which divided into inhibition and sterilization steps. The optical density measurement, malondialdehyde concentration detection, morphologic imaging with electronic microscopy and Fourier transform infrared spectroscopic analysis unveiled the interaction of Ag2O NPs with E. coli, which verified the inhibition-sterilization process we proposed.