Medicinal plant-associated fungi called endophytes may have a role in promoting plant growth via multiple pathways. More people are becoming aware of endophytic fungi as sources of bioactive substances with beneficial medicinal properties. In the present study, two endopytic fungi were isolated from two medicinal plants, Ocimum basilicum and Ammi visanga, and were identified as Alternaria alternata AUMC16440 and Curvularia pseudobrachyspora AUMC16441. Petroleum ether and ethyl acetate were used to extract secondary metabolites from these fungi. The petroleum ether and ethyl acetate extracts of these endophytic fungi exhibit potent antimicrobial activity against human pathogenic microbes such as Escherichia coli, Bacillus subtilis, Aspergillus chrysogenium, and Aspergillus fumigatus. Moreover, Curvularia pseudobrachyspora AUMC16441 and Alternaria alternata AUMC16440 were able to produce indole acetic acid with concentrations of 9.62 and 5.7 µg/mL, respectively, when tested with 5 µg/mL tryptophan and had antidiabetic activities with percentages of 94% and 82%, respectively. The bioactive substances of the fungal and plant extracts were identified using gas chromatography-mass spectrometry analysis (GC-MS) that revealed the presence of different compounds belonging to different groups, such as fatty acids, flavonoids, hydrocarbons, phenolics and alcohols. The most relevant compounds were hexadecanoic acid, tetradecane, phenol 2, 2'-methylenebis [6-(1, 1-dimethylethyl)-4-methyl, 13-docosenamide, 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, ethyl iso-allocholate, cyclooctasiloxane hexadecamethyl, cycloheptasiloxane tetradecamethyl, and cyclohexasiloxane dodecamethyl. These substances are recognized to have a number of advantageous biological characteristics. In addition, the GC-MS result revealed that many compounds are common between endophytic fungal isolates and their host, confirming the role of endophytic fungi in protecting their host and vice versa.