The bactericidal effects of nanomaterials play an essential role in cytoplasmic leakage, leading to bacterial cell death. In this study, silver nanoparticles (AgNPs) were synthesized using a fungal extract of Aspergillus fumigatus (A. fumigatus). The physicochemical properties of the bare and myco-synthesized AgNPs (MS-AgNPs) were examined by X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, ultraviolet–visible (UV–vis) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and transmission electron microscopy (TEM). XRD revealed the crystalline structure of the prepared NPs. The FTIR spectrum of the MS-AgNPs revealed the presence of the stretching vibrations of hydroxyl (−OH) and carbonyl groups (C=O). The UV results showed absorption from 450 nm to 590 nm, confirming the synthesis of the AgNPs. SEM and TEM showed rough cubic shapes (spheres), 20–60 nm in size, while EDX confirmed the presence of 60% Ag in the sample. The MS-AgNPs revealed the highest antibacterial activity against Staphylococcus aureus, with a zone of inhibition of 18.21 ± 2.1 mm, followed by Shigella dysenteriae and Salmonella typhi. The bimetallic-AgNPs played a vital role in cell membrane damage and the release of cellular contents, specifically nucleic acids and proteins. These results suggest that MS-AgNPs have promising antimicrobial capabilities and might be beneficial for an extensive array of biological applications.