The biochemical behavior (biomass production, accumulation of total lipid, substrate uptake, fatty acid composition of fungal oil) of two oleaginous Mucorales strains, namely Mortierella isabellina ATHUM 2935 and Cunninghamella echinulata ATHUM 4411, was studied when the aforementioned microorganisms were cultivated on xylose, raw glycerol and glucose under nitrogen-limited conditions. Significant differences in the process of lipid accumulation as related to the carbon sources used were observed for both microorganisms. These differences were attributed to the different metabolic pathways involved in the assimilation of the above substrates. Therefore, the various carbon sources were channeled, at different extent, to storage lipid or to lipid-free biomass formation. Although glucose containing media favored the production of mycelial mass (15gL−1 of total biomass in the case of C. echinulata and 27gL−1 in the case of M. isabellina), the accumulated lipid in dry matter was 46.0% for C. echinulata and 44.6% for M. isabellina. Lipid accumulation was induced on xylose containing media (M. isabellina accumulated 65.5% and C. echinulata 57.7% of lipid, wtwt−1, in dry mycelial mass). In these conditions, lipids of C. echinulata contained significant quantities of γ-linolenic acid (GLA). This fungus, when cultivated on xylose, produced 6.7gL−1 of single cell oil and 1119mgL−1 of GLA. Finally, the growth of both C. echinulata and M. isabellina on raw glycerol resulted in lower yields in terms of both biomass and oil produced than the growth on xylose.