The utilization of appropriate preparations of plant growth-promoting fungi (PGPF) is one of the most promising trends for inducing plant resistance against a pathogen. However, not much is known about their mechanisms. Herein, the goal of this study was to present more information on this matter. The isolated rhizosphere fungi Aspergillus falvus, Aspergillus niger, Penicillium citrinum, Penicillium chrysogenum, and Trichoderma koningiopsis were tested for plant growth promotion traits. In Triticum aestivum L., root colonizing PGPF stimulated induced systemic resistance (ISR) against wilt disease by Rhizoctonia solani R43, resulting in a restriction of symptoms and disease development. This study demonstrates that the PGPF strains activated the pathogenesis-related gene (PR-1, 2), plant defensive chitinase (Chit-1) and β-1, 3-glucanase (Glu-2) genes and increased the plant-specific defensive proteins against Rhizoctonia solani pathogen. Their ability for ISR stimulation in T. aestivum L. was investigated in comparison to benzothiadiazole (BTH) as a chemical inducer. PGPF treatments showed overexpression of the defensive genes and consequently fewer disease symptoms compared to the chemical inducer BTH and control. Physical damage of lesions by fungal pathogen was less severe in leaves treated with PGPF strains compared to control plants. The observation of smaller lesion widths in the leaves pre-treated with the PGPF strains may be due to induced expression of protein genes, which is described as ISR. Quantitative real-time PCR (qRT-PCR) investigation showed that PR-1.2, Chi-1, and, Glu-2 genes were remarkably activated in PGPF-treated wheat plants.