The diversity of fungal endophytes in Sorghum bicolor was investigated in samples collected from 10 different geographical regions of Karnataka state, India. A total of 360 endophytes were isolated from leaf, stem, and root tissues and were assigned to 26 fungal species based on morphology and molecular characterization using ITS sequences. All the endophytes belonged to the phylum Ascomycota. The diversity (Shannon H, 2.57; Simpson_1-D, 0.92) and species richness (Margalef's, 4.68; Menhinick, 3.61) were found to be higher for the endophytes isolated from leaf tissues. The species evenness of the endophytic assemblage was strongly influenced by tissue type, followed by geographical location. The biocontrol potential of isolated endophytes was evaluated against economically destructive sorghum grain mould pathogens such as Fusarium thapsinum, Epicoccum sorghinum, Alternaria alternata, and Curvularia lunata using the dual culture method. Biocontrol potential was exhibited by 26 endophytic isolates, of which Trichoderma asperellum recorded broad-spectrum activity against target pathogens, followed by E. nigrum and A. longipes. Most (82%) endophytes showed plant growth-promoting traits. Biosynthesis of indole-3-acetic acid (IAA) was observed in 84% of isolates, and phosphate solubilization, siderophore production, and cellulase activity was observed in 69%, 23%, and 27% of isolates, respectively. Seeds treated with T. asperellum exhibited a significantly higher seed vigour index (2096), germination percentage (94%), and yield under greenhouse and field conditions. The results were substantiated by the confocal microscopy analysis, which clearly demonstrated the colonization of treated endophyte in root tissues. The present study reveals an ecofriendly approach to explore T. asperellum in sorghum disease management.