Microbial oil is a potential substitute for vegetable oils in the biodiesel industry. Efforts to obtain cheap carbon sources for the cultivation of lipid-producing microorganisms comprise an active research area. This work aimed to extract the hemicellulose fraction from Eucalyptus uograndis and to use its hydrolysate as a carbon source for Rhodotorula toruloides (an oleaginous yeast) cultivation for microbial oil production. Hemicellulose hydrothermal extractions were performed at different temperatures, times, and ratios of solid to liquid (S/L). Temperature and time showed a stronger effect on the solubilization of hemicellulose. Hemicellulose extraction at 155 °C, 195 min, and an S/L ratio of 1/2 resulted in a hydrolysate with a xylose content of 37.0 g/l. R. toruloides cultivation in this hydrolysate showed that initial pH had a strong influence on cell growth. At an initial pH of 6.2, cells grew to 6.0 g/l of biomass with a lipid content of 50%. Therefore, we believe that E. urograndis hemicellulose hydrolysate could be a potential substrate for R. toruloides for lipid production based on the biorefinery concept.