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Background: Lentil (Lens culinaris Medik.) is one of the major pulse crops in the world. The present study aimed to evaluate the effect of Trichoderma isolates to control the charcoal rot disease caused by Macrophomina pseudophaseolina recently reported on lentil plants. Results: In this study, antagonistic effects of seven isolates of Trichoderma spp. isolated from the rhizosphere of lentil were evaluated in vitro against Macrophomina pseudophaseolina M1. All isolates of Trichoderma significantly reduced the mycelial growth of M1 in vitro. Maximum reduction of the pathogen growth was observed at T10 (72.53%), followed by T5 (63.7%) and T6 (62.88%), while T2 caused the minimum reduction (37.33%). Likewise, Trichoderma isolates significantly reduced the size and number microsclerotia. M1 produced the minimum number of microsclerotia in the presence of T10, followed by T1 causing reduction of 68.56 and 53.37% over control, respectively. The maximum reduction of microsclerotia’s size was observed in the presence of T10 (54.92%), followed by T1 (49.84) and T8 (48.68%). Results also revealed that production of volatile metabolite, components and inhibition of the test pathogen by volatile metabolites varied among different antagonistic Trichoderma. T1 produced the most volatile compounds, followed by T2 and T3 and T10 causing mycelial growth inhibition of 41.66, 33.88, 30.55 and 30.37%, respectively. The efficacy of the antagonistic biocontrol agents on charcoal rot was evaluated in vivo. Seed treatment with T10 significantly protected lentil seedlings from M. pseudophaseolina as compared to untreated plants and also improved their growth. T10 isolate was identified based on molecular tools, using internal transcribed spaces and Translation elongation factor of ribosomal DNA. The results identified T10 as Trichoderma longibrachiatum with accession number MW818102 and OK500004 deposited in NCBI GenBank database. Conclusion: The study revealed the first report of using Trichoderma longibrachiatum as a promising biological agent against M. pseudophaseolina causing charcoal rot disease of lentil.