Squalene and docosahexaenoic acid (DHA) have gained substantial market share as dietary supplements and vital nutraceuticals due to their beneficial effects on human health. Marine fish are the main commercial source of these nutraceuticals, but a growing global demand, issues of sustainability, and an expanding vegan and vegetarian population has prompted the search for alternatives. Oils obtained from oleaginous microorganisms such as microalgae, diatoms, certain fungi, and thraustochytrids are alternatives to fish oils for omega-3 fatty acids. Among these, DHA is now being mined from thraustochytrids due to its highest proportion in their lipids, however, this strategy is not cost-effective. One way to offset such elevated production costs is to simultaneously extract other high value-added biological products from these oleaginous microorganisms. Here, we propose a novel biorefinery process based on single-step purification of squalene from total lipids extracted from an oleaginous thraustochytrid cultivated on non-edible forest biomass. To render the process economically feasible and sustainable, additional squalene-free lipids were exploited for enrichment of DHA; whereas leftover lipids generated as by-product during the process were tested as a novel biolubricant.