The emergence of antimicrobial resistance in foodborne bacterial pathogens has raised significant concerns in the food industry. This study explores the antimicrobial potential of biosynthesized silver nanoparticles (AgNPs) derived from Agaricus bisporus (Mushroom) against foodborne bacterial pathogens. The biosynthesized AgNPs were characterized using various techniques, including UV-visible spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, high-resolution scanning electron microscopy with energy dispersive X-ray spectroscopy, dynamic light scattering, and zeta potential analysis. The antibacterial activity of the AgNPs was tested against a panel of foodborne bacterial strains, and their cytotoxicity was evaluated on normal human skin fibroblasts. Among the tested strains, Pseudomonas aeruginosa ATCC 27853 showed the highest sensitivity with an inhibition zone diameter (IZD) of 48 mm, while Klebsiella quasipneumoniae ATTC 700603 and Bacillus cereus ATCC 11778 displayed the highest resistance with IZDs of 20 mm. The silver cations released by AgNPs demonstrated strong bactericidal effects against both Gram-positive (G + ve) and Gram-negative (G - ve) bacteria, as evidenced by the minimum inhibitory concentration/minimum bactericidal concentration (MBC/MIC) ratio. Moreover, cytotoxicity testing on normal human skin fibroblasts (HSF) indicated that AgNPs derived from the mushroom extract were safe, with a cell viability of 98.2%. Therefore, AgNPs hold promise as an alternative means to inhibit biofilm formation in the food industry sector.