The goal of the current work was to investigate the antibacterial, antibiofilm, anticancer, and antioxidant opportunities of silver and gold nanoparticles (AgNPs and AuNPs) synthesized utilizing a new fungus strain called Fusarium pseudonygamai TB-13c. With the aid of UV, HR-TEM, FTIR, SEM, and XRD, the NPs’ creation was examined. For AgNPs and AuNPs, the mycosynthesized NPs’ highest peak plasmon band was seen at around 420 and 540 nm, respectively. AgNPs ranged in size from 5 to 20 nm, whereas AuNPs ranged in size from 8 to 60 nm. AgNPs and AuNPs were spherical in form. For AgNPs, the angles were 38.42°, 44.56°, 64.66°, and 77.75°; for AuNPs, the angles were 38.56°, 44.74°, 64.87°, and 77.85°. The antibacterial efficiency of AgNPs and AuNPs was evaluated against Klebsiella pneumoniae, Pseudomonas aeruginosa, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus aureus (MRSA). In particular, AgNPs outperformed AuNPs in their ability to combat pathogenic microorganisms. Furthermore, antibiofilm study that shown AuNPs had activity more than AgNPs. Interestingly, applying the DPPH procedure these noble metallic NPs had antioxidant activity, which the IC50 for AgNPs was 38.2 μg/ mL and 180 μg/ mL for AuNPs. The modification in the cells was evident in the cytotoxicity evaluation findings as change of their usual shape, partially or completely loss of monolayer, granulation, and shrinkage, or cell rounding with IC50 for normal vero cell were 695.34 μg/mL and 631.66 μg/mL for AgNPs and AuNPs, respectively, whilst IC50 for cancer cell (Mcf7) was 204.07 μg/mL for AgNPs and 206.95 μg/mL for AuNPs. The combined results show that there is a clear and flexible biological use for less toxic chemotherapeutic agents.