Conventional microalgae oil extraction applies physicochemical destruction of dry cell biomass prior to transesterification process to produce fatty acid methyl esters (FAMEs). This report presents a simple and rapid direct transesterification (DT) method for FAMEs production and fatty acid profiling of microalgae using freshly harvested biomass. Results revealed that the FAMEs recovered from Chlorella vulgaris were 50.1 and 68.3 mg with conventional oil-extraction-transesterification (OET) and DT method, respectively. While for Messastrum gracile, the FAMEs recovered, were 49.9 and 76.3 mg, respectively with OET and DT methods. This demonstrated that the DT method increased FAMEs recovery by 36.4% and 53.0% from C. vulgaris and M. gracile, respectively, as compared to OET method. Additionally, the DT method recovered a significantly higher amount of palmitic (C16:0) and stearic (C18:0) acids from both species, which indicated the important role of these fatty acids in the membranes of cells and organelles. The DT method performed very well using a small volume (5 mL) of fresh biomass coupled with a shorter reaction time (~ 15 min), thus making real-time monitoring of FAMEs and fatty acid accumulation in microalgae culture feasible.