Fresh pomegranate peel extract was employed to synthesize silver oxide nanoparticles (Ag₂O NPs). Rapid formation of stable Ag₂O NPs was observed on exposure to the aqueous fresh pomegranate peel extract with solution of AgNO₃. The Ag₂O NPs were characterized by X-ray analysis, scanning electron microscopy (SEM), ultraviolet–visible (UV–Vis) spectroscopy, and Fourier transform infrared spectroscopy (FTIR). The X-ray diffraction (XRD) confirmed that the forming Ag₂O NP has a crystalline size of 37 nm, while SEM micrographs revealed a comparatively spherical shape, with the size of ~ 64 nm. The Ag₂O spectrum displayed a peak in the visible range and a blue shift at 461 nm corresponding to the Plasmon absorbance of silver nanoparticles. Four bacterial strains and one type of fungus were tested using Ag₂O NPs. The results showed the negative influence of Ag₂O NPs on the growth rate, thus implying the significance of the present study in production of biomedical products.