The most suitable approach for manufacturing different metal and metal oxide nanoparticles is to use fungi. The purpose of this work is to examine how fungi that have been isolated from soil produce CuO-nanoparticles (CuO-NPs). It has been demonstrated that Aspergillus niger (A. niger), which was obtained from soil and recognized using both conventional and molecular methods, is capable of producing CuO- nanoparticles. UV, XRD, FITR, and TEM have all been used to characterize these nanoparticles. The antioxidant potential of synthesized CuO-nanoparticles was examined by the process of DPPH scavenging. Red blood cells' membrane stabilization technique was used to investigate the anti-inflammatory properties. The MTT procedure was utilized to explore the anticancer activity. The antiviral was identified by the decrease in the ability of Coxsackie-B, Adenovirus-22, and Adenovirus-40 to infect Vero Cells. CuO nanoparticles have a spherical form, a peak at 345 nm, and diameters in a range from 4 to 18 nm. Using the DPPH technique, the biosynthesized CuO-NPs were found to have antioxidant action, with an IC₅₀ value of 27.93±2.4 µg/mL. The anti-inflammatory proportion of the biosynthesized CuO-NPs was 98.6% at 1000 µg/mL and dropped to 48.9% at 100 µg/mL. The biosynthesized CuO-NPs exhibited anticancer action versus MCF-7, PANC1, and Caco2 cells, with IC₅₀ values of 82.02 ± 1.04 µg/mL, 91.45 ± 1.89, and 151.45 ± 0.89, respectively. Ascending concentrations of CuO-NPs enhanced their antiviral effect (%) against the Co-B4, Adeno-22 and Adeno-40 viruses. The myco-synthesized CuO-NPs showed encouraging results and have the potential to be utilized in a range of therapeutic uses.