Plant-based nanoparticles (NPs) have many advantages over physical and chemical methods and featured with several medicinal and biological applications. In this study, zinc oxide NPs (ZnO-NPs) were synthesized using pomegranate peel aqueous extract, under mild and ecofriendly conditions. The ZnO-NPs structure, morphology, and optical properties were investigated using X-ray diffraction (XRD), scanning electron microscope (SEM), Fourier transform infrared (FTIR), and ultraviolet-visible (UV-Vis). Antibacterial activity against Gram-positive and Gram-negative strains were evaluated using the disk diffusion method. The effect of extract concentration (20, 30, and 40 mL) on the final properties of NPs, as well as the NPs concentration used for antibacterial test (50, 100, and 200 mg/mL), were also studied. The results indicate a hexagonal structure with particle size increases as extract concentration increase (D = 18.53, 29.88, and 30.34 nm), while the optical bandgap was decreased (Eg = 2.87, 2.80, and 1.92 eV). The antibacterial activity of ZnO-NPs indicated high efficiency, similar or even higher than that of the control azithromycin, more against S. aureus, increased with NPs concentration, and preferred when NPs prepared from high extract concentration. Such promising physicochemical properties support the usefulness and efficacy of the reported bio-route for production of ZnO-NPs and may encourage its application for large-scale production.