The green synthesis of silver nanoparticles (SNPs) is the most economical, rapid, facile, and eco-friendly technique. In this study, pathogenic bacteria from human stool were identified, and the antibacterial potency of Nigella sativa L. seed extracts containing SNPs was assessed. The identification of the isolated pathogenic bacteria was achieved through the analysis of their 16S rDNA sequences. The characterization of SNPs was done by ultraviolet–visible spectroscopy (UV-VIS spectroscopy), scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). Six pathogenic bacteria were isolated from human stool and isolated bacteria were identified by 16S rDNA sequencing analysis. SNPs were found in the size range of 30-60nm and showed good antibacterial and antifungal activity. The maximum zone of inhibition was found against A. spanius at 32.0±0.8nm. Achromobacter spanius, Pseudomonas geniculate, and Enterobacter asburiae WL115 were extremely sensitive to heated treated nanoparticles at 100μgml-1. In the current work, N. sativa L. was investigated because it produced SNPs effectively, may be used as a safe, affordable agent, and may have antibacterial and antifungal properties. The potential biological uses of SNPs will be encouraged by these discoveries, which will drive future research.