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BACKGROUND Nanomaterials have recently been identified for their potential benefits in the areas of medicine and pharmaceuticals. Among these nanomaterials, silver nanoparticles (Ag-NPs) have been widely utilized in the fields of diagnostics, antimicrobials, and catalysis. OBJECTIVE To investigate the potential utility of Citrobacter freundii in the synthesis of silver nanoparticles (Ag-NPs), and to determine the antimicrobial activities of the Ag-NPs produced. METHODS Aqueous Ag+ ions were reduced when exposed to C. freundii extract and sunlight, leading to formation of AgNPs. Qualitative microanalysis for the synthesized Ag-NPs was done using UV-vis spectrometry, energy dispersive X-ray analysis (EDX), and scanning and transmission electron microscopy. The hydrodynamic size and stability of the particles were detected using dynamic light scattering (DLS) analysis. The Ag-NPs' anti-planktonic and anti-biofilm activities against Staphylococcus aureus and Pseudomonas aeruginosa, which are two important skin and wound pathogens, were investigated. The cytotoxicity on human dermal fibroblast cell line was also determined. RESULTS Ag-NPs were spherical with a size range between 15 to 30 nm. Furthermore, Ag-NPs displayed potent bactericidal activities against both S. aureus and P. aeruginosa and showed noticeable anti-biofilm activity against S. aureus biofilms. Ag-NPs induced minor cytotoxic effects on human cells as indicated by a reduction of cell viability, a disruption of plasma membrane integrity, and apoptosis induction. CONCLUSION Ag-NPs generated in this study might be a future potential alternative to be used as antimicrobial agents in pharmaceutical applications for wound and skin related infections.