The antibacterial effect of ZnO nanoparticles is tested against Staphylococcus aureus, (a Gram-positive pathogenic bacterium) from a particle-size, concentration, and surface-defects point of view. Activation of antibacterial activity was achieved by standard well diffusion agar and minimum inhibitory concentration procedures. Our results show that smaller-sized particles are more effective inhibitors of bacterial activity when used in a certain optimum concentration. To reveal the underlying mechanism of the observed size and concentration-dependent bacterial activity inhibition, we measured the concentrations of Zn2+ ions released in each suspension by an inductive couple plasma optical emission spectrophotometer. Additionally, photoluminance spectra of our samples show significant surface defects (mainly oxygen vacancies) that generate reactive oxygen species. The underlying mechanism of the observed size- and concentration-dependent bacterial activity inhibition is attributed primarily to the release of Zn2+ ions and generation of reactive oxygen species that interact and penetrate the cell membrane, causing lethal damage to the cell. Finally, the antibacterial effectiveness and maximum sensitivity of our nanoparticles is confirmed by optical density measurements.