The present work deals with three methods proposed for the direct transesterification of lipids in the filamentous fungi Aspergillus niger and in the oleaginous yeast Yarrowia lipolytica, grown on distillery spent wash (DSW). For comparison, the reported amount of fatty acid methyl esters (FAMEs) generated by the improved methods applied to dry and wet biomass was normalized against the amount of FAMEs yielded via a two-step extraction/methanolysis method from the same samples. To increase the esterification reactions yield from wet biomass, modifications of the process were evaluated, including: the use of a co-solvent; hydrolysis pretreatment; and the use of a double catalyst. The methylated derivatives generated by each method were then analyzed by gas chromatography with flame ionization detector (GC-FID). It is shown that direct transesterification yielded higher amounts of FAMEs than the two-step methods in both microorganisms, with normalized FAMEs productions of: 146 % (A. niger) and 190 % (Y. lipolytica) from dry biomass, and 119 % (A. niger) and 143 % (Y. lipolytica) from wet biomass, respectively. In the case of wet Y. lipolytica, the double catalyst improvement approach generated 20 % higher FAMEs production than the direct acid methanolysis of the same material. The contributions of the methods parameters for improved esterification and for the FAMEs production profile for Y. lipolytica are also discussed.