Ultrasonic oscillation and nanometer grind were used to treat ZnO suspension, and the effects of treatment time on preparation of ZnO were investigated. PEG400 was chosen as a suitable surfactant to reduce aggregation problem because PEG400 is not only effective at keeping the stability of ZnO suspension, but also has no influences on diameter size of ZnO nanoparticles or antibacterial ability. The effects of concentration and particle size on antibacterial ability of ZnO suspension were evaluated. The results demonstrated that the optimum time of ultrasonic oscillation for reducing aggregation is 0.5 h, and diameter size exhibited linear decline during the first 1 h of nanometer grind. Antibacterial ability increased with the increasing of the concentration of ZnO nano-suspension until it reached 0.25 g/L, and antibacterial ability was inversely proportional to diameter size of ZnO nanoparticles.