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In this study, we investigated the synthesis of silver nanoparticles using Peganum harmala water extract at ambient temperature. The Ag nanoparticles (AgNPs) were characterized by ultraviolet–visible spectroscopy (UV–vis), Fourier transform infrared spectroscopy (FT–IR), powder X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), and transmission electron microscopy (TEM). The average particle size of the silver nanoparticles was about 23 nm. Inhibitory activity of the synthesized AgNPs was tested against human pathogens like Escherichia coli and Staphylococcus aureus. The results indicated that the AgNPs showed moderate inhibitory actions, demonstrating its antibacterial value against pathogenic diseases.