ZnO-based nanomaterials have high antifungal effects, such as inhibition of growth and reproduction of some pathogenic fungi, such as Fusarium sp., Rhizoctonia solani and Macrophomina phaseolina. Therefore, we report the extracellular synthesis of ZnONPs using a potential fungal antagonist (Trichoderma harzianum). ZnONPs were then characterized for their size, shape, charge and composition by visual analysis, UV–visible spectrometry, X-ray diffraction (XRD), Zeta potential, transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDX). The TEM test confirmed that the size of the produced ZnONPs was 8–23 nm. The green synthesized ZnONPs were characterized by Fourier transform infrared spectroscopy (FTIR) studies to reveal the functional group attributed to the formation of ZnONPs. For the first time, trichogenic ZnONPs were shown to have fungicidal action against three soil–cotton pathogenic fungi in the laboratory and greenhouse. An antifungal examination was used to evaluate the bioactivity of the mycogenic ZnONPs in addition to two chemical fungicides (Moncut and Maxim XL) against three soil-borne pathogens, including Fusarium sp., Rhizoctonia solani and Macrophomina phaseolina. The findings of this study show a novel fungicidal activity in in vitro assay for complete inhibition of fungal growth of tested plant pathogenic fungi, as well as a considerable reduction in cotton seedling disease symptoms under greenhouse conditions. The formulation of a trichogenic ZnONPs form was found to increase its antifungal effect significantly. Finally, the utilization of biocontrol agents, such as T. harzianum, could be a safe strategy for the synthesis of a medium-scale of ZnONPs and employ it for fungal disease control in cotton.