The shift toward sustainable biofuels and bioproducts has increased interest in microbial production systems using renewable substrates. This study explores the use of wood hydrolysate, an abundant, cost-effective lignocellulosic substrate, as the primary carbon source for lipid and carotenoid production by Rhodosporidium toruloides-7191 under fed-batch cultivation in a 3-L bioreactor. The fed-batch strategy, chosen over batch and continuous modes, enables controlled nutrient supply, minimizes substrate inhibition, and maintains a favorable carbon-to-nitrogen ratio, thereby supporting prolonged biosynthesis and higher product yields. The process achieved a maximum lipid production of 22.33 g/L, a total lipid accumulation of 57.9% and a total carotenoid production of 4.23 mg/L. Fatty acid analysis shows a composition rich in linoleic acid (C18:2), oleic acid (C18:1), and palmitic acid (C16:0), indicating its suitability for biodiesel applications. The results emphasize R. toruloides-7191 as a promising candidate for industrial-scale applications, advancing sustainable production of biofuels and high-value bioproducts. The effectiveness of wood hydrolysate as a substrate further supports the feasibility of this approach, highlighting its potential in advancing industrial-scale processes for the production of biofuels and value-added compounds.