Aims of this research were to produce biodiesel from lipid of an endophytic fungus and to investigate the influence of cultivation parameters on lipid production. It was found that time, amount of malt extract, carbon source and nitrogen source affected the lipid production and %unsaturation of triglyceride. The maximum lipid production was at the beginning of stationary phase of the growth. The lipid, produced by the fungus from all cultivation conditions, comprised of about 12% free fatty acid (FFA) while the unsaturated fatty acid component of the lipid depended on the cultivation conditions. The fungus, cultured in MEB, produced the lipid with 53% unsaturated fatty acid. The suitable method for biodiesel production was produced by a two-stage transesterification process. The first stage was acid pretreatment with 1% H₂SO₄, which could reduce the FFA level of lipid of endophytic fungus to less than 1%. The second stage, alkali base catylyze transesterification using 1% NaOH, gave 94.78% of methyl ester content and 97.34% of conversion.