This study investigates the production of zinc oxide nanoparticles (ZnO NPs) using Aspergillus niger culture filtrates as a sustainable and environmentally friendly approach, combining them with a zinc carbonate solution. The produced ZnO nanoparticles were examined using transmission electron microscopy (TEM), energy dispersive X-ray diffraction (EDX), scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). The characterization data validated the creation of highly crystalline ZnO NPs with an average size range from 27 to 40 nm. ZnO NPs effect on A. ochraceus and A. niger growth at ideal temperatures was investigated. At doses of 0.25%, 0.5%, and 1%, respectively, Aspergillus niger and A. ochraceus caused 56%, 81%, and 87% and 64%, 71%, and 86% of the inhibition of fungal growth, respectively. At the highest ZnO NPs concentration, the maximum inhibition rate was observed. This research highlights the potential of Aspergillus niger as a bio-factory to produce ZnO nanoparticles with promising applications in agriculture and other fields. The eco-friendly synthesis method, coupled with the antifungal properties of the synthesized ZnO nanoparticles, provides a sustainable and environmentally friendly alternative to conventional fungicides for plant disease management.