A study was conducted to understand the diversity of endophytic fungi from various natural hosts and their antimicrobial and alkali-tolerance activity with bioactive metabolite profiling and in-silico docking. A total of 176 endophytic fungi were isolated from nine hosts from India, and classified into 31 genera based on their morphology and molecular analysis of the ITS rDNA gene region. The endophytic community was dominated by fungi belonging to the genus Colletotrichum (20.5%), followed by Nigrospora (14.03%), Pestalotiopsis (9.35%), Lasiodiplodia (8.2%) and lastly by Hypoxylon (7.01%). The diversity of endophytic fungal species was highest in Catharanthus roseus, whereas it was lowest in Piper longum. Representative endophytic fungi were screened for their antimicrobial activity against seven clinical pathogens. Nearly 77% of the isolates showed potential antimicrobial activity against one or more test pathogens. Interestingly, Xylaria sp. and Nigrospora sphaerica showed robust antimicrobial activity compared to the standard antibiotics. Further, the alkali tolerance demonstrated that nearly 30% of the isolates grew well at high pH. Two of the isolates (Fusarium equiseti CR-10 and Colletotrichum fructicola GS-10) showed remarkable growth in alkaline media compared to standard media. Further, Xylaria sp. metabolites were characterized by Liquid Chromatography-Mass Spectrometer (LC-MS). In-silico docking analysis revealed compounds like substance P, geldanamycin, 1- palmitoyl- 2 -linoleoyl-sn-glycero- 3 -phosphate, 7,7- dimethyl-(5Z,8Z)- eicosadienoic acid, 6-3-(1-Adamantyl)- 4-hydroxyphenyl- 2-naphthalenecarboxylic acid and 15-ketoiloprost was found to exhibit potent antibacterial activity with higher binding energy with selected bacterial proteins compared to standards. This suggests that this endophytic fungal metabolite can be a potential source for producing promising natural antimicrobial drugs.