Due to the overexploitation of industrial fisheries, as the principal source of fish oil, as well as the increasing replacement of synthetic pigments for animal nutrition, we need to find sustainable sources for these essential nutrient productions. Marine Rhodotorula strains NCYC4007 and NCYC1146 were used to determine the biosynthesis of docosahexaenoic acid (DHA) and carotenoids by biotransforming raw glycerol, a waste product of biodiesel. To evaluate the presence of inhibitory substances in raw glycerol, both strains were also grown in the presence of analytical grade glycerol and glucose as the main carbon source separately. With raw glycerol, NCYC4007 showed a significant correlation between DHA production and intracellular phosphorous concentrations. NCYC1146, a new Rhodotorula strain genetically described in this work, can produce canthaxanthin but only when glycerol is used as a main carbon source. Then, NCYC4007 could synthesize DHA as a phospholipid, and the production of canthaxanthin depends on the kind of carbon source used by NCYC1146. Finally, malate dehydrogenase activity and glucose production can be used as a proxy of the metabolisms in these marine Rhodotorula. This is the first evidence that marine Rhodotorula are capable of synthesizing DHA and canthaxanthin using an alternative and low-cost source of carbon to potentially scale their sustainable production for animal nutrition.