The production of H2O2 has been taken for a crucial reason for antimicrobial activity of ZnO without light irradiation. However, how the H2O2 generates in ZnO suspension is not clear. In the present work, the comparatively detections on three kinds of ZnO, tetrapod-like ZnO whiskers (t-ZnO), nanosized ZnO particles (n-ZnO), and microsized ZnO particles (m-ZnO), showed that the antimicrobial activity of ZnO was correlated with its production of H2O2. Oxygen vacancy (V-O) in the surface layer of ZnO crystals determined by XPS indicated that it was quite probably involved in the production of H2O2. To validate the role of Vo, the concentration of V-O in t-ZnO was adjusted by heat-treatment under the atmospheres of H-2, vacuum, and O-2, respectively, and the H2O2 production and antimicrobial effect were detected. Consistently, the t-ZnO treated in H-2, which possessed the most Vo in its crystal, produced the most H2O2 and displayed the best antimicrobial activity. These results provide the basis for developing a more detailed mechanism for H2O2 generation catalyzed by ZnO and for taking greater advantage of this type of antimicrobial agent.