Fusarium root rot disease of tomato caused by Fusarium solani is one of the most important tomato diseases and causes severe problems in all production regions. The aim of the current study was to evaluate the efficacy of some Trichoderma isolates and Pseudomonas fluorescence to control this disease in vitro and in vivo. In laboratory experiments, the effect of four Trichoderma isolates, i.e. T. harzianum (T₁), T. harzianum (T₂), T. reesei and T. asperellum (T₃₄) and one isolate P. fluorescence. Data indicated that all the tested isolates of biocontrol agents were able to inhibit the mycelial growth of the pathogen with variation in their antagonistic capability. In general, T. asperellum (T₃₄)exhibited the highest antagonistic effect toward the pathogen, followed by T. harzianum (T₂). Under greenhouse conditions, application of bio control agents as a soil treatment led to decrease in disease severity of tomato root rot disease compared to the control treatment. Data indicated that T. reesei treatment exhibited the highest reduction in the disease severity of root rot compared to other treatments in both seasons. Moreover, application of these treatments significantly enhanced plant height (cm), fresh and dry weight (gm). Laboratory estimates showed that the tested bio control agents increased catalase (m/g fresh weight), peroxidase and polyphenol oxidase (m/g f. w) in treated tomato plants compared to infected and healthy control. Furthermore, treated tomato plants contained a high level of total phenol contents (TPC) compared to untreated plants.