Charcoal rot is the most important fungal disease on soybean in Iran. According to the absence of the resistant cultivars, soil inhabitant and the high pathogenicity rate of the pathogen, chemical and crop control alone is not succeed. To improve the efficiencyof disease biological control on soybean in greenhouse, the wild type Trichoderma koningii NAS-K1 (non radiated) and the selective mutant NAS-K1M25 isolates were used. The induction of mutation in wild type increases the biological control efficiency of the disease. The efficiency of powder, granular and seed coating bio-formulations, were evaluated separately in greenhouse. Powder has the best effect in reducing the disease; granular bio-formulation has the best effect on plant growth and its yield and seed coating, has the best effect on seed germination characteristics and vigor index. The application of these bio-formulations increased soybean growth indices compared to the control and chemical treatments. The highest yield production increase in the presence of the pathogen was observed by seed coating treated with NAS-K1M25. Study on viability of biological agent in powder and granular bio-formulations, showed that it has slight decrease and still effective for a period of 11 months at room temperature, but keeping it at 4 °C has a positive effect on it.