The production of zinc oxide nanoparticles (ZnO NPs) by fungi is developing as a critical nanotechnology topic because of its green, eco-friendly, non-toxic, and cost-effective nature. Fusarium oxysporum. has been used as a stabilizing and reducing agent in the biosynthesis of ZnO NPs. Various characterization approaches, including UV-Vis spectroscopy and X-ray diffraction (XRD), confirmed the synthesis of ZnO NPs with an average size of 26.3 nm. Furthermore, the micrographs of transmission electron microscopy (TEM) revealed that ZnO NPs had a crystalline structure with hexagonal wurtzite from 5 to 47 nm size. Fourier transform infrared (FTIR) pattern, revealed the presence of reducing and stabilizing agents such as carboxylic acid, and amines. The antibacterial activity of ZnO NPs against Gram-positive and Gram-negative clinical bacterial isolates was remarkable where ZnO NPs have shown strong antibacterial activity against Salmonella typhi more than Escherichia coli, whereas, at a dosage of 50 mg/ mL, ZnO NPs had slightly more activity against Bacillus subtilis than Staphylococcus aureus.