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The infection of Aspergillus flavus and its aflatoxin production pose a severe threat to humans, animals as well as plants life. Their inhibition using green techniques are considered as one of the important challenges. The present study outlines the antifungal activity of the ZnO nanoparticles (NPs) synthesized from lemongrass leaf extract and their effect on the mycelial growth of Aspergillus flavus and its aflatoxins production. The qualitative and quantitative analyses of aflatoxins were determined, respectively using thin-layer chromatography and spectrophotometric methods. The X-ray diffraction as well as scanning and transmission electron microscopy studies indicated the formations of hexagonal ZnO NPs having the sizes ranged between 7 and 14 nm. FTIR spectrum confirmed the formation of ZnO NPs. The ZnO NPs displayed 92.25% inhibition of the growth of A. flavus and 100% inhibition of the aflatoxins production at the concentrations of 200 μl/mL and 150 μl/mL respectively. The present biosynthetic method is a simple, cost-effective, eco-friendly, high yield, green and handy protocol capable of synthesizing ZnO NPs, which might have accomplished due to the activities of plant metabolites and phytochemicals available in the lemongrass leaves parenchyma. This study revealed that ZnO NPs have the potential to forbid the growth of A. flavus and its aflatoxins production. Hence, ZnO NPs could be used in the plant protection and as a preservative for safe storage of food commodities to prevent A. flavus contamination and aflatoxins poisoning in coming future. [ABSTRACT FROM AUTHOR]