Background: Silver nanoparticles (AgNPs) have recently been extensively investigated because of their superior physical, chemical, and biological characteristics, and their superiority is primarily due to the size, shape, composition, crystallinity, and structure of AgNPs compared to their bulk forms. Objective: The current study aimed to investigate the antibacterial activity of silver nanoparticles (AgNPs) synthesized intracellular by using standard strain Bacillus subitilis ATCC 6633 against reference strains Escherichia coli ATCC 2592 and Staphylococcus aureus ATCC 29737. Results: The synthesized AgNPs showed potent antibacterial activities against the two tested bacterial strains with inhibition zones ranged from 42 -52mm and MIC 27.2 μg / ml. The silver nanoparticles were characterized with particle size ≃ 100 nm and zeta potential -19. There was deformation in both tested strains upon treatment with AgNPs which was observed by Scanning Electron Microscopy (SEM). Conclusion: The results indicated that AgNPs could be used as an effective antibacterial agent.