Rhizoctonia solani causes severe diseases in many plant species, particularly root rot in tomato plants. For the first time, Trichoderma pubescens effectively controls R. solani in vitro and in vivo. R. solani strain R11 was identified using the ITS region (OP456527); meanwhile, T. pubescens strain Tp21 was characterized by the ITS region (OP456528) and two genes (tef-1 and rpb2). The antagonistic dual culture method revealed that T. pubescens had a high activity of 76.93% in vitro. A substantial increase in root length, plant height, shoot fresh and dry, and root fresh and dry weight was indicated after applying T. pubescens to tomato plants in vivo. Additionally, it significantly increased the chlorophyll content and total phenolic compounds. The treatment with T. pubescens exhibited a low disease index (DI, 16.00%) without significant differences with Uniform® fungicide at a concentration of 1 ppm (14.67%), while the R. solani-infected plants showed a DI of 78.67%. At 15 days after inoculation, promising increases in the relative expression levels of three defense-related genes (PAL, CHS, and HQT) were observed in all T. pubescens treated plants compared with the non-treated plants. Plants treated with T. pubescens alone showed the highest expression value, with relative transcriptional levels of PAL, CHS, and HQT that were 2.72-, 4.44-, and 3.72-fold higher in comparison with control plants, respectively. The two treatments of T. pubescens exhibited increasing antioxidant enzyme production (POX, SOD, PPO, and CAT), while high MDA and H2O2 levels were observed in the infected plants. The HPLC results of the leaf extract showed a fluctuation in polyphenolic compound content. T. pubescens application alone or for treating plant pathogen infection showed elevated phenolic acids such as chlorogenic and coumaric acids. Therefore, the ability of T. pubescens to inhibit the growth of R. solani, enhance the development of tomato plants, and induce systemic resistance supports the application of T. pubescens as a potential bioagent for managing root rot disease and productivity increase of crops.