Nanotechnology holds great potential in advanced water and wastewater treatment to improve treatment efficiency. Zinc oxide nanoparticles (ZnO NPs) have received considerable attention due to their unique antibacterial activities toward various microorganisms that are commonly found in the environment. In the present study, ZnO NPs were synthesized through both mechano-chemical and sol–gel methods. The synthesized ZnO NPs were characterized through X-ray diffraction and transmission electron microscopy techniques. Then, their antibacterial activities against separated wastewater bacteria were evaluated by determining the zone inhibitor, the minimum inhibitory concentration, and the minimum bactericidal concentration. The results were compared with those obtained from wastewater after chlorine disinfection and ultraviolet (UV) disinfection. These studies demonstrated that the antibacterial activity of ZnO NPs depends on the type and the strain of bacteria. They have also demonstrated that the activity increases as the concentration of ZnO NPs increases. Overall, the experimental results suggest that ZnO NPs can potentially be an antibacterial reagent to treat wastewater. They can particularly be applied as a complementary method with UV disinfection. Thus, they can be developed as antibacterial agents to improve wastewater quality.