Nanotechnology has developed as an emerging field and modern research with potential biomedical sciences. Emerging of new infectious diseases and the occurrence of drug resistance bacteria is challenging to the researchers. This would initiate the search for unique constituent to be predictable new antimicrobials. Extracellular biosynthesis of silver nanoparticles (Ag-NPs) was carried out by Fusarium oxysporum (MTCC-2480). The colour change of culture supernatant from pale to brown colour indicates the reduction of silver metals into silver ions. Silver nanoparticles were further characterized and confirmed by performing thin layer chromatography (TLC). A single separate band of silver nanoparticle was observed by TLC with Rf value of 0.75. Silver nanoparticles have been evaluated for antibacterial activities against selected human pathogens by disc and well method. Silver nanoparticle exhibited highest antibacterial activity against Salmonella typhi followed by Pseudomonas aeroginosa. Silver nanoparticles were subjected to study the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against the selected human pathogens. MIC revealed that 20μl/ml of Ag-NPs exhibited highest inhibition activity against Klebsiella pneumoniae followed by Pseudomonas aeruginosa and MBC revealed that 100% inhibition was observed against Pseudomonas aeruginosa. The nanobiotechnological fugal based biosynthesis of silver nanoparticles has high potential as antibacterial bio-resource.