The filamentous fungi Trichoderma is used asmost important biocontrol agent. Because of the absence of sexual reproduction in this fungus, other methods of genetic improvement have been developed to enhance its bicontrol potential. In the present investigation protoplast fusion of four different Trichoderma spp. was evaluated for their antagonistic activity against the soil borne plant pathogen like Fusarium spp. which causes wilting diseases in many crops. In protoplast fusion technique, protoplast isolation by lysis enzyme at the concentration of 10 mg/ml, protoplasts were fused intra and inter specifically, with 30% (w/v) PEG solution and those fusants regenerated on osmatic stabilizer amended PDA media. Eight self fusants and eighteen fusants were selected for assessment of antagonastic activity against fusarium spp. in dual culture technique. The results revealed that among eight intra fusants, fusantTvdsf-2 (93.70%) was recorded highest per cent inhibition against F. o. f. sp. udumandTvssf-1 (86.70%) against the F. solani and inter specific fusants, the maximum per cent inhibition mycelia growth was observed in Tvs×Tas-1 (94.07%) and Tvd×Tas-1 (94.07%) against F. o. f. sp. udumand Tvd×Tas-1 (89.63%) against the F. solani. These outcomes indicated that protoplast fusion technique was successful tools to enhance the antagonistic effects of Trichoderma species against several fungal plant pathogens.