Abstract: Colletotrichum sp. have potential to act as antidiabetic agent, due to its α-glucosidase inhibitory. Therefore, the objective of present study was to isolate and identify the bioactive compounds responsible for the α-glucosidase inhibitory activity in Colletotrichum sp. TSC13. The methanol extract of TSC13 mycelia, was partitioned with n-hexane, chloroform and ethyl acetate. The n-hexane fraction exhibited the strongest α-glucosidase inhibitory activity. Column chromatography of this fraction resulted in 8 sub-fractions (F1-8). Fraction 3 (F3) which showed 71.4±2.4% inhibition was analysed further. Analysis using GC-MS after methylation of F3 and comparison to spectra databases and confirmation using authentic sample standards showed that F3 had two saturated fatty acid methyl esters, palmitic acid and stearic acid methyl esters and three unsaturated fatty acid methyl esters, oleic acid, linoleic acid and linoleinic acid methyl esters. Unsaturated fatty acids showed higher activity than the saturated fatty acids and the methyl esters form of unsaturated fatty acids showed slightly less active than the free acids. Further analysis using an ethyl acetate extract, it was confirmed that most of the fatty acids were present in the form of free acids. Therefore, it was concluded that the α-glucosidase inhibitor compounds in Colletotrichum sp. TSC13 were unsaturated fatty acids. This is the first report that a Colletotrichum sp. from T. sumatrana has α-glucosidase inhibitory activity.