Abstarct: Bean is an industrial and strategic crop, which is one of the most important and widely consumed legumes in the world, especially for the poor population. Bean root rot disease caused by Fusarium species is an important and common disease of bean, which annually destroys a high percentage of this valuable resource. Considering the health and environmental side effects of using chemical pesticides, alternative methods such as biological control were applied in the recent years. In this study, the antagonistic activity of Trichoderma lixii and its mutant isolates in controlling F. equiseti, F. acuminatum and F. solani pathogens were investigated in vitro and in vivo. This study showed the possibility of improving mycelia growth rate, the antagonistic capability, extracellular proteins and enzymes production for biological control of plant diseases through mutation with γ-radiation. Among the studied Trichoderma wild type strains, T. lixii (T20) had the highest ability to inhibit growth of all three pathogenic species, which was selected for mutation induction. Also, the mutation improved mycelial growth at 48 hours, antagonistic activity, sporulation, spore and conidia size, phialide arrangement compared to the wild type. Mutant isolates had higher chitinase activity. The SDS-PAGE profiles showed several enzyme bands such as cellobiohydrolases, endoglucanases, β-glucosidases, endochitinases and β-(1, 4)-N-acetyl glucoaminidases. Also, DNA fingerprinting using PAPD-PCR marker and OPA10, OPA11 and OPA16 primers, showed significant genetic diversity between mutants and wild type strain, and the combination of three primers was able to differentiate mutants and wild type at the similarity level of 65%. Finaly the results of greenhouse showed that treatment with Trichoderma mutants could reduce the disease severity (AUDPC) compared to the other treatments significantly. Plants treated with Trichoderma (mutants and wild type) showed a significant increase in the content of protein, chlorophyll a, b, carotenoid, PPO enzyme, total phenol, peroxidase and SOD. In contrast, the lowest level of MDA was observed in these plants.