Four lipid extraction methods (Bligh & Dyer, hexane & isopropanol, dichloromethane & methanol, and hexane) were evaluated to extract lipid from freeze- and oven-dried fungus Mortierella isabellina ATCC42613. The highest lipid yield (41.8%) was obtained from Bligh & Dyer extraction on the oven-dried fungal biomass with a methanol:chloroform:water ratio of 2:1:0.8. Other lipid extraction methods on both freeze- and oven-dried samples had lipid yields ranging from 20.7% to 35.9%. Non-polar lipid was the main lipid class (more than 90% of total lipid) in M. isabellina. Regarding fatty acid profile, there was no significant difference on fatty acid concentration between different drying and extraction meth- ods. Estimation of biodiesel fuel properties using correlative models further demonstrated that the fungal biodiesel is a good alternative to fossil diesel.