The rationale of the present study was to isolate and identify endophytic fungi from endemic medicinal plants in Eastern Ghats and screened for antimicrobial potential of isolated fungal crude extracts. A total of 329 endophytic strains were isolated from 600 infected leaves and stem cuttings of endemic plants. The diversity and species richness was analyzed statistically and found to be higher in leaf segments than in stem segments. From isolated fungal strains, was identified using molecular identification methods and selected as the most potent plumbagin-producing endophytic strain. Further the isolation and structural characterization of endophytic fungal plumbagin (5-hydroxyl-2-methylnaptalene-1,4-dione) was purified and confirmed through spectroscopy analysis. The molecular weight was determined as 188 in positive mode by ESI-MS, which confirmed to be plumbagin which potentially inhibited all tested pathogens, therefore the endophytic fungal plumbagin from the current study possesses important biological activities against pathogens.