In light of climatical variations, fungal diseases increased, which led to heavy losses in economic crops. As a result of these concerns, the management of phytopathogenic fungi has emerged to be one of the most important global issues. Green-synthesized nanomaterials have a remarkable antimicrobial efficacy to be used as an alternate to harmful fungicides. Herein, this study focused on establishing the prospective effects of silver nanoparticles (AgNPs) synthesized by Aspergillus ustus ON076464 against phytopathogenic F. oxysporum to control wilt disease of pepper plant. The biosynthesized AgNPs were subjected to different characterization practices. The results evoked the ability of Aspergillus ustus filtrate to build up AgNPs in a spherical shape and dispersed without aggregation with a size average of 12.4 nm. Laser diffraction revealed that the particles obtained were monodispersed in a mixture having an average diameter of 17.20 nm. It is conceivable from the results that AgNPs at 100 µg/ml and 50 µg/ml are markedly effective against F. oxysporum exhibiting the highest antifungal action and minimum inhibitory concentration MIC respectively. The results of the scanning electron microscope (SEM) of F. oxysporum mycelium treated with AgNPs showed severe morphological destruction when compared to the nontreated hyphae. AgNPs at concentrations (50 and 25 µg/ml) highly gave a reduction in percent disease index by (25 and 37.5) and highly gave protection percent by (72.7 and 59.06 %) compared to the untreated infected plants. The application of AgNPs at concentrations (50 and 25 µg/ml) resulted in different responses regarding the photosynthetic pigments, total carbohydrates content, phenol, and total protein of Fusarium-infected plants. Consequently, this investigation presents a promising insight into fungi for AgNPs biosynthesis, and the application of these nanoparticles could effectively limit Fusarium wilt disease of pepper plants, as a biological and novel approach.