In the current study, zinc oxide nanoparticles (ZnO-NP) were prepared using extracellular extracts of Aspergillus niger. Hence, the morphological structure, optical, and surface features of the synthesized nanoparticles were studied by X-ray diffraction, transmission electron microscopy, ultraviolet-visible and infrared absorption by Fourier transform. Use dynamic light scattering and zeta potential measurements to assess colloidal stability. The mean size of the synthetic particles is approximately 20 ± 5 nm and they have a hexagonal crystal structure. In addition, the prepared nanoparticles have strong light absorption in the ultraviolet region of λ = 265 and 370 nm. To achieve the goal of this study, the efficiency of ZnO-NP was determined as an antibacterial and antifungal against different bacterial and fungal strains. It was found that ZnO-NP showed significant antibacterial activity, where the inhibition zones were varied from 21 to 35mm in diameter against six bacterial species (i.e. K. pneumoniae, E. coli, A. baumannii, P. aeruginosa, S. aureus, and S. haemolyticus). In such a case, the minimal inhibitory concentration of zinc oxide nanoparticles against bacterial strains were 50, 12.5, 12.5, 50, 12.5, and 12.5μg/ml for K. pneumoniae, E. coli, A. baumannii, P. aeruginosa, S. aureus, and S. haemolyticus, respectively. Furthermore, ZnO-NP exhibits an antifungal behaviour against four fungal species (i.e., A. niger, P. marneffei, C. glabrata, and C. parapsilosis) with inhibition zone from 18 to 35mm in diameter. Whereas, the MICs for fungal isolates were 12.5μg/ml except A. niger was at 25μg/ml. Wi-38 cells were treated with ZnO-NPs exhibited different levels of cytotoxicity dependent upon the concentration of ZnO NPs using the MTT assay with IC₅₀~800.42. Therefore, the present study introduces a facile and cost-effective extracellular green-synthesis of ZnO-NP to be used as antimicrobial and anticancer agents.