Nowadays, scientists are making serious efforts to enhance the antibacterial and anticancer activity of ZnO nanostructures and to understand their mechanisms of action. In this study, different strategies were conducted to synthesize ZnO-based nanostructures with improved activities, including positively and negatively-charged functionalized ZnO nanoparticles (using polyethyleneimine and 3-mercaptopropionic acid, respectively), doped ZnO particles (Ni-doped ZnO), and two nanocomposites (ZnO/chitosan/Ag2O and ZnO/Ag/GO). Physicochemical properties, colloidal stabilities, and the activity of all samples against MCF-7 cancer cells and S. aureus and E. coli bacteria were studied. Additionally, the effect of the strategies on the two main mechanisms of action of ZnO, ROS generation and Zn2+ ion release, was studied to address the current uncertainty in the dominant mechanism. Overall, making a nanocomposite using graphene oxide (GO) and introducing a positively charged surface were the most effective strategies to enhance the ZnO activity. Moreover, the results showed that doping of ZnO with metal ions is a superior approach for enhancing the antibacterial activity even in dark conditions. An increase in intracellular ROS levels, the negligible concentration of Zn2+ ions and no correlation between the sample's activity and the released Zn2+ ions indicated the dominant role of non-dissolved particles and ROS generation on the particle surfaces.