In the present study, 51 fungal endophytes (FEs) were isolated, purified and identified from the healthy leaf segments of ten grapevine varieties based on the spore and colony morphologies and ITS sequence information. The FEs belonged to the Ascomycota division comprising eight genera viz., Alternaria, Aspergillus, Bipolaris, Curvularia, Daldinia, Exserohilum, Fusarium and Nigrospora. The in vitro direct confrontation assay against Colletotrichum gloeosporioides revealed that six isolates viz., VR8 (70%), SB2 (83.15%), CS2 (88.42%), MN3 (88.42%), MS5 (78.94%) and MS15 (78.94%) inhibited the mycelial growth of test pathogen. The remaining 45 fungal isolates showed 20-59.9% growth inhibition of C. gloeosporioides. Indirect confrontation assay manifested that the isolates MN1 and MN4a showed 79.09% and 78.18% growth inhibition of C. gloeosporioides followed by MM4 (73.63%) and S5 (71.81%) isolates. Isolate S5 and MM4 were found to produce azulene and 1,3-Cyclopentanedione, 4,4-dimethyl as antimicrobial volatile organic compounds, respectively. The 38 FEs showed PCR amplification using internal transcribed spacer universal primers. The BLAST search revealed highest similarity with the existing sequences in the database. The phylogenetic analysis revealed the occurrence of seven distinct clusters each corresponding to single genus. The online version contains supplementary material available at 10.1007/s13205-023-03675-z.