To tackle the proliferation of pathogenic microorganisms without relying on antibiotics, innovative materials boasting antimicrobial properties have been engineered. This study focuses on the development of graphene oxide/silver (GO/Ag) nanocomposites, derived from partially reduced graphene oxide adorned with silver nanoparticles. Various nanocomposites with different amounts of silver (GO/Ag-1, GO/Ag-2, GO/Ag-3, and GO/ Ag-4) were synthesized, and their antibacterial efficacy was systematically studied. The silver nanoparticles were uniformly deposited on the partially reduced graphene oxide surface, exhibiting spherical morphologies with an average size of 25 nm. The nanocomposites displayed potent antibacterial properties against both gram-positive bacteria (S. aureus and B. subtilis) and gram-negative bacteria (E. coli and S. enterica) as confirmed by minimum inhibition concentration (MIC) studies and time-dependent experiments. The optimal MIC for Gram-positive bacteria was 62.5 μg/mL and for Gram-negative bacteria was 125 μg/mL for the GO/Ag nanocomposites. Bacterial cells that encountered the nanocomposite films exhibited significantly greater inhibitory effects compared to those exposed to conventional antibacterial materials. Furthermore, the cytotoxicity of these nanocomposites was assessed using human epithelial cells (HEC), revealing that GO/Ag-1 and GO/Ag-2 exhibited lower toxicity levels toward HEC and remained compatible even at higher dilution rates. This study underscores the potential of GO/Ag-based nanocomposites as versatile materials for antibacterial applications, particularly as biocompatible wound dressings, offering promising prospects for wound healing and infection control.