Environmental pollution due to the improper use of the chemical fungicides represents a vital ecological problem, which affects human and animal health, as well as the microbial biodiversity and abundance in the soil. In this study, an endophytic fungus Aspergillus oryzae YRA3, isolated from the wild plant Atractylis carduus (Forssk.) C.Chr, was tested for its biocontrol activity against Rhizoctonia root rot of sorghum. The antagonistic potential of A. oryzae YRA3 was tested against Rhizoctonia solani in vitro. A full inhibition in the growth of R. solani was recorded indicating a strong antagonistic potential for this endophyte. To investigate the chemical composition of its metabolites, GC/MS analysis was used and thirty-two compounds in its culture filtrate were identified. Among these metabolites, some compounds with an antifungal background were detected including palmitic acid, 2-heptanone, and 2,3-butanediol. To these antifungal metabolites the antagonistic activity of A. oryzae YRA3 can be attributed. In the greenhouse experiment, treating of the infected sorghum plants with A. oryzae YRA3 significantly reduced severity of the Rhizoctonia root rot by 73.4%. An upregulation of the defensive genes (JERF3), (POD) and (CHI II) was recorded in sorghum roots when were inoculated with A. oryzae YRA3. In addition, an increment in the activity of peroxidase and polyphenol oxidase, as well as the total phenolic content in the sorghum roots was also recorded. Furthermore, the results obtained from the greenhouse experiment revealed a growth-promoting effect for inoculating the sorghum plants with A. oryzae YRA3. It can be concluded that A. oryzae YRA3 can be a probable biological agent to control this disease in sorghum. However, its evaluation under field conditions is highly needed in the future studies.