Multidrug resistance bacteria (MDR) are a serious and increasing public health problem. For controlling pathogen growth, new strategies such as biosynthesized nanoparticles by Fusarium oxysporum extract are urgently needed. The biosynthesis of silver nanoparticles (AgNPs) as an antibacterial agent against MDR clinical isolates bacteria such as Staphylococcus aureus and Klebsiella pneumonia was determined. Characterization of AgNPs by UV–Vis, SEM, EDAX, TEM, and SAED, Zeta potential, and antibacterial activity was performed. UV–Vis spectrum of the aqueous medium containing AgNPs showed an absorption peak at around 438 nm. TEM images detected the spherical AgNPs shape with a size ranging from (6.51-58 nm). SAED pattern study confirmed the crystalline structure of AgNPs. In this study, synthesized AgNPs exhibited strong antibacterial activity against MDR (S. aureus and k. pneumonia). Protein banding profile and RAPD PCR of S. aureus and k. pneumonia using different RAPD primers showed a strong effect of AgNPs on the genome of the tested bacteria. In conclusion; AgNPs could be an effective antibacterial agent against MRD bacteria.