Garlic (Allium sativum L.) being infected by mycotoxigenic fungi is one of the primary factors limiting its nutritional and medical value. Therefore, there is an urgent need to repress mycotoxigenic fungi utilizing safer treatments, possibly involving nanoparticles. Fusarium incarnatum was isolated from garlic (A. sativum L.) that showed fungal contamination and their identification was confirmed using amplified and sequenced internal transcribed spacer nuclear ribosomal DNA regions, which confirmed the isolation of F. incarnatum from all cultivars. Copper oxide nanoparticles (CuONPs) at different concentrations showed inhibitory activity against F. incarnatum growth and mycotoxins, particularly at 400 ppm. The production of F. incarnatum mycotoxins, i.e., beauvericins, fusarins, moniliformin, and enniatins, was inhibited to 62.8%, 45.4%, 58.1%, and 55.0%, respectively at 400 ppm of CuONPs compared to the control. Shrinkage of the F. incarnatum cell membrane and collapsing of the cell walls were recorded via transmission electron microscopy at 400 ppm, but negligible distortion appeared at 100 ppm of CuONPs. CuONPs at 100 ppm encouraged the activity of CMC-ase, zylanase, and amylase, while 200 and 400 ppm promoted less enzyme activity. The current findings suggest that CuONPs have a fungistatic effect on F. incarnatum and their mycotoxins.