This study evaluated the suitability of pinewood pyrolysates as a carbon source for lipid production and cultivation of the oleaginous yeast Rhodosporidium diobovatum and the microalgae Chlorella vulgaris. Thermal decomposition of pinewood and fractional condensation were used to obtain an oil rich in levoglucosan which was upgraded to glucose by acid hydrolysis. Blending of pyrolytic sugars with pure glucose in both nitrogen rich and nitrogen limited conditions was studied for R. diobovatum, and under nitrogen limited conditions for C. vulgaris. Glucose consumption rate decreased with increasing proportions of pyrolytic sugars increasing cultivation time. While R. diobovatum was capable of growth in 100% (v/v) pyrolytic sugars, C. vulgaris growth declined rapidly in blends greater than 20% (v/v) until no growth was detected in blends >40%. Finally, the effects of pyrolysis sugars on lipid composition was evaluated and biodiesel fuel properties were estimated based on the lipid profiles.