Zinc oxide (ZnO) nanoparticles possess antibacterial properties. Being less toxic to humans than silver, they are attractive as antibacterial agents in biomedical applications. In this study, we focus on the influence of the size of ZnO nanoparticles on their antibacterial action against strains of three bacteria: one Gram-negative, Escherichia coli and two Gram-positive, Bacillus subtilis and Staphylococcus aureus. The antibacterial efficacy of the nanoparticles increases with decreasing particle size. A major contributor to antibacterial action is the oxidative stress induced by the ZnO. To understand the relationship between antibacterial action and induced oxidative stress, we measured the dependence of the nanoparticle diameter on the hydrogen peroxide concentration. Even at a fixed nanoparticle concentration, the hydrogen peroxide concentration increased with decreasing nanoparticle diameter. This is qualitatively similar to the dependence of the antibacterial activity on the nanoparticle diameter. In addition, in the presence of ZnO nanoparticles, we detected increased quantities of endogenous hydrogen peroxide in the E. coli. For use as antibacterial wound dressings, we fabricated nonwoven fiber mats from poly(vinyl alcohol) (PVA)/ZnO nanoparticle suspensions. The antibacterial efficacy of the PVA/ZnO electrospun fiber mats also increased with a decrease in the diameter and an increase in the concentration of the ZnO nanoparticles.