In recent years, microbial platforms for biofuels and nutraceuticals have gained recognition as sustainable alternatives. This study investigates titanium dioxide nanoparticles’ effect on lipid and omega fatty acid production in Thraustochytrium sp. Both titanium dioxide and nitrogen starvation independently enhance lipid content, with a synergistic effect observed when combined. Titanium dioxide increases lipid content by 37.69% (up to 78.84%), while nitrogen starvation achieves a 34.91% enhancement (total lipid 75.92 ± 2.43%). The combined approach yields the highest lipid content at 81.48 ± 3.14%, denoting a 46.19% increase. Additionally, titanium dioxide increases omega-6 (LA by 66.95%) and omega-9 (OA by 44.98%) levels, while nitrogen starvation enhances omega-3 (mainly EPA, by 91.83%) levels. This eco-friendly strategy has implications for bioenergy and nutraceuticals, offering a cost-effective means of enhancing oleaginous microorganisms for biodiesel production. The research outcomes contribute to addressing key obstacles in cost-efficient biodiesel production, impacting the economic landscape of the biodiesel industry.