Microbial nanoparticles (NPs) have become the subject of immense research interest in the recent past due to their wide range of applications as effective antimicrobial agents, drug delivery systems, gene delivery, diagnostic agents in imaging diseases, and consumer products among others. The present study emphasizes on the synthesis of metallic silver NPs (SNPs) from cell-free supernatant of Aspergillus awamori strain KGSR12. The phase purity, composition, size, and shape of the as-synthesized NPs were characterized using various analytic spectroscopic techniques including X-ray diffraction, scanning electron microscope (SEM), ultraviolet-visible, and Fourier transform-infrared spectroscopy. Based on the SEM analysis, the particles are uniformly distributed, and size is estimated to be 40-50 nm. Antibacterial activity of NPs against significant human pathogens was conferred with well diffusion assay, and it reveals the strains of Pseudomonas aeruginosa, Klebsiella pneumoniae, and Bacillus cereus are susceptible to synthesized SNPs that confirm the antibacterial activity of SNPs. Thus, the study concludes with the biogenic and eco-friendly route for synthesizing SNPs with antibacterial activity against clinically important pathogens and attributes growing interest on fungi as an emerging source for the synthesis of NPs.