The active α-glucosidase inhibitor compounds in the endophytic fungus Colletotrichum sp. TSC13 were found to be the unsaturated fatty acids (oleic, linoleic and linolenic acids). These compounds have potential as antidiabetic agents. The aim of the present study is to investigate the effects of various media composition on growth (mycelium dry weight) and the fatty acids content (μg mg(-1) mycelium DW) of Colletotrichum sp. TSC13 in relation to its α-glucosidase inhibitory activity. For that purpose, the experiments were set up by varying the carbon and nitrogen sources, metal ions and desaturase and fatty acid synthase inhibitors in the media. Colletotrichum sp. TSC13 grown on potato dextrose broth (PDB) was used as control. The α-glucosidase inhibitory activities were (range from 43.9 ± 2.5 to 88.6 ± 5.2%) at 10 μg mL(-1). This activity seemed to correlate with the unsaturated fatty acids content of the samples. Different sugars as carbon source experiment showed that xylose gave the highest growth (938.7 ± 141.6 mg). However, the highest fatty acids content was obtained from fructose medium which containing linoleic acid (38.8 ± 4.9 μ g mg(-1) DW). Soluble starch gave better growth (672.5 ± 62.3 mg) but very low fatty acids content (2.8 ± 0.1 μg mg(-1) DW) was obtained. Yeast extract was the best nitrogen source. Fatty acids production was better as compared to beef extract and soytone. This is the first report of various media compositions on fatty acids content in Colletotrichum sp. TSC13 in relation to its α-glucosidase inhibitory activity.