The present study was aimed to green synthesis and characterization of zinc oxide nanoparticles (ZnONPs) from Aspergillus niger, which was evaluated for their antioxidant, antimicrobial and anticancer activity. The synthesised NPs were characterized by various analytical techniques such as UV–VIS Spectroscopy, FT-IR, XRD, DLS, SEM, and TEM. It was confirmed through the UV–Vis spectrophotometer; corresponding peaks were identified at 390 nm. The green synthesised ZnONPs were characterized by FT-IR studies to reveal the functional group attributed to the formation of ZnONPs. Morphological size of ZnONPs was 80–130 nm found through characterization by DLS, SEM, and TEM. Furthermore, the green synthesised ZnONPs showed potent antioxidant (ABTS and DPPH assay) antimicrobial activity against human pathogenic bacterial strains such as Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Enterobacter aerogenes. In addition, the green synthesised ZnONPs showed the dose-dependent cytotoxicity and apoptotic features in human hepatocellular carcinoma cells (HepG2). The overall findings of the study suggested that A. niger had a potential for the biosynthesis of ZnONPs as an alternative biomaterial for future therapeutic application as an antioxidant, antimicrobial and anticancer compound.