Flower-shaped zinc oxide (ZnO) nanostructures were prepared via a simple aqueous precipitation strategy at room temperature. The as-grown nanostructures were characterized by UV–vis spectroscopy, UV–vis diffuse reflectance spectroscopy (DRS), spectrofluorometry, Fourier transform infrared (FTIR) spectroscopy with attenuated total reflection (ATR), X-ray diffraction (XRD), and field emission scanning electron microscopy (FESEM). The antifungal and anti-aflatoxigenic activities of the ZnO nanostructures were further investigated using a highly toxigenic strain of Aspergillus flavus Link under in vitro and in situ conditions. The results showed that the A. flavus isolate was inhibited to various extents by different concentrations of ZnO nanostructures, but the best inhibitions occurred at 1.25, 2.5, and 5 mM in the culture media. At these concentrations, suppression of aflatoxin biosynthesis (99.7%) was also observed. Moreover, a reasonable reduction in the aflatoxin content (69%) was observed in maize grains treated with the lowest ZnO concentration that exhibited the strongest inhibitory activity in the liquid media. SEM micrographs clearly indicate multiple degenerative alterations in fungal morphology after treatment with ZnO such as damage of the tubular filaments, loss of hyphae shape, as well as hyphae rupture. These results suggest that flower-shaped ZnO nanostructures exhibit strong antifungal and anti-aflatoxigenic activity with potential applications in the agro-food system.