A worldwide hazard to human health is posed by the growth of toxic bacteria that have contaminated fresh, processed, cereal, and seed products in storage facilities. As the number of multidrug-resistant pathogenic microorganisms rises, we must find safe, and effective antimicrobials. The use of green synthesis nanoparticles to combat microbial pathogens has gained a rising interest. The current study showed Aspergillus fumigatus was applied as a promising biomass for the green synthesis of biogenic silver nanoparticles (Ag NPs). The UV-visible spectra of biosynthesized Ag NPs appeared characteristic surface plasmon absorption at 475 nm, round-shaped with sizes ranging from 17.11 to 75.54 nm and an average size of 50.37 ± 2.3 nm. In vitro tests were conducted to evaluate the antibacterial, antioxidant, and anticancer effects of various treatment procedures for Ag NP applications. The synthesized Ag NPs was revealed biological activity against Aspergillus flauvas, A. niger, Bacillus cereus, Candida albicans, Esherichia coli, Pseudomonas aerugonosa, and Staphylococcus aureus under optimum conditions. The test bacteria were sensitive to low Ag NPs concentrations. (5, 10, 11, 8, 7, 10, and 7 mg/mL) was observed for the mentioned-before tested microorganisms, respectively. The bacterial pathogens described above experienced their biofilm formation effectively suppressed by Ag NPs at sub-inhibitory doses. Ag NPs were tested using scanning electron microscopy (SEM) to verify their antibacterial efficacy towards S. aureus and P. aeruginosa. These findings clearly show how harmful Ag NPs are to pathogenic bacteria. The Ag NPs showed antitumor activity with IC50 at 5 μg/mL against human HepG-2 and MCF-7 cellular carcinoma cells, while 50 mg/mL was required to induce 70% of normal Vero cell mortality. These findings imply that green synthetic Ag NPs can be used on cancer cell lines in vitro for anticancer effect beside their potential as a lethal factor against some pathogenic microbes.