Industrial production of biodiesel from microbial catalysts requires large volume of low-cost feedstock for lipid production. Vinasse, also known as distillery spent wash (DSW), is a liquid waste produced in large amounts by ethanol distilleries. This effluent is particularly rich in organic matter, and may be considered as a potential resource for the production of fungal lipids. The present study aimed at evaluating the potential of vinasse from a distillery located in Reunion Island for yeast and fungal growth, lipid production, and suitability for biodiesel requirements. Among the 28 different strains tested, we found that Aspergillus niger grown on pure vinasse allowed biomass production of up to 24.05 g/L (dry weight), whereas Aspergillus awamori produced the maximum amount of lipid, at 2.27 g/L. Nutrient removal and vinasse remediation were found to be the best for A. niger and Cryptococcus curvatus, reaching a maximum of 50% for nitrogen, and A. awamori showed 50% carbon removal. Lipids produced were principally composed of C16:0, C18:1 (n-9), and C18:2 (n-6), thus resembling the vegetal oil used in the biodiesel production. This work has shown that vinasse can support production of biomass and lipids from fungi and yeast suitable for energetic use and that its polluting charge can be significantly reduced through this process.