The biorefinery approach ensures a sustainable source of valuable fatty acids and opens up new avenues for their application in healthcare industries. Recent studies highlight the health benefits of omega-PUFAs, spurring the search for cost-effective production methods. Microbial platforms are promising for high-yield PUFA production, with ω-3 dominating the market. ω-3 PUFAs offer antioxidant and anti-inflammatory effects, reducing illness risk, while all PUFAs contribute to cardiovascular health, diabetes prevention, cancer risk reduction, and more. ω-6 PUFAs, particularly linoleic acid (LA) and arachidonic acid (ARA), play vital roles in various aspects of health, making them high-demand bioavailable compounds. Additionally, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exhibit potential benefits in brain development and COVID-19 prevention. This comprehensive review provides insights into the state-of-the-art microbial biorefinery strategies for ω-3 and ω-6 PUFA production and their wide-ranging health-related benefits.