A systematic and complete antibacterial study on well-designed and well-characterized microparticle (micro), nanoparticle (nano), and capped nano ZnO has been carried out in both dark and light conditions with the objective of arriving at the mechanism of the antibacterial activity of ZnO, particularly in the dark. The present systematic study has conclusively proved that reactive oxygen species (ROS) such as •OH, •O2-, and H2O2 are significantly produced from aqueous suspension of ZnO even in the dark and are mainly responsible for the activity in the dark up to 17%, rather than Zn2+ ion leaching as proposed earlier. This work further confirms that surface defects play a major role in the production of ROS both in the presence and absence of light. In the dark, superoxide (•O2-) radical mediated ROS generation through singly ionized oxygen vacancy is proposed for the first time, and it is confirmed by EPR and scavenger studies. ROS such as •O2-, H2O2, and •OH have been estimated by UV-visible spectroscopy using nitro blue tetrazolium (NBT), KMnO4 titrations, and fluorescence spectroscopy, respectively. These are correlated to the antibacterial activity of ZnO in the dark and light. The activity is found to be highest for nano ZnO and least for micro ZnO, with capped ZnO between the two, highlighting the important role of surface defects in generation of ROS. The surface charge density of ZnO in dark and light has been estimated for the first time to the best of our knowledge, and it can influence antibacterial activity. Our work proposes a new mechanism mediated by superoxide species, for antibacterial activity of ZnO especially in the dark. © 2015 American Chemical Society.