Producing biofuel from microalgae has gained renewed interest recently. Schizochytrium limacinum is a heterotrophic microalga that is capable of producing high levels of biomass and total fatty acid. The objective of this work is to explore the potential of producing biodiesel fuel from this alga using different biodiesel preparation methods, including oil extraction followed by transesterification (a two-stage method) or direction transesterification of algal biomass (a one-stage method). When freeze-dried biomass was used as feedstock, the two-stage method resulted in 57% of crude biodiesel yield (based on algal biomass) with a fatty acid methyl ester (FAME) content of 66.37%. The one-stage method (with chloroform, hexane, or petroleum ether used in transesterification) led to a high yield of crude biodiesel, whereas only chloroform-based transesterification led to a high FAME content. When wet biomass was used as feedstock, the one-stage method resulted in a much-lower biodiesel yield. The biodiesel prepared via the direct transesterification of dry biomass was subjected to ASTM standard tests. Parameters such as free glycerol, total glycerol, acid number, soap content, corrosiveness to copper, flash point, viscosity, and particulate matter met the ASTM standards, while the water and sediment content, as well as the sulfur content did not pass the standard. Collectively, the results indicate the alga S. limanicum is a suitable feedstock for producing biodiesel via the direct transesterification method.