SILVER nanoparticles (AgNPs) are broadly applied in numerous industries due to their exclusive physico-chemical and antimicrobial properties. Herein, the biosynthesis of extremely stable silver nanoparticles by the extracellular extract of the novel strain Aspergillus sakultaensis AUMC13885 is investigated for the first time. The physico-chemical characteristics of the synthesized AgNPs were assayed by using UV–vis spectroscopy, transmission electron microscopy (TEM) and Fourier transform infrared spectrometry (FT-IR). The UV-vis recorded a maximum absorption band at 405nm, which matched to the surface plasmon absorbance of silver nanoparticles. Aspergillus sakultaensis AUMC13885 synthesized a uniformly distributed AgNPs of 5-25nm in size estimated by transmission electron microscopy (TEM). Detection of the proteins binding to the synthesized nanoparticles was conducted by FT-IR analysis. The optimum conditions for AgNPs biosynthesis in this study were 1.0mM substrate, alkaline pH, reaction temperature of 80°C, and reaction time of 120hrs. The activity of AgNPs against human pathogenic bacteria was conducted by well diffusion assay. Bacillus cereus, Staphylococcus aureus, Klebsiella pneumoniae, and Proteus vulgaris were sensitive to the synthesized AgNPs that affirm the antibacterial activity. Consequently, this study contributes with the eco-friendly biogenic way for producing potential antibacterial AgNPs against resistant human pathogens and attributes growing interest on fungi as a sustainable source for nanoparticles synthesis.