Omega fatty acids, major importance in the prevention or treatment of a range of human diseases or disorders related with inflammation. These fatty acids are found in transgenic plants, fungi, and animals and even in microorganisms but in major amounts can be extracted from fatty fish. However, due to bioaccumulation of fat-soluble vitamins and high levels of saturated and omega-6 fatty acids, they may have deleterious health effects. It becomes necessary to search for novel and rich sources containing omega-3 fatty acids and one of the alternatives include fungi. The present study deals with production and purification of omega-3 fatty acids from Trichoderma viride and Aspergillus niger. In the present study, the main objective was to explore the beneficial effects of fungi for the maximum lipid production through optimized conditions and the results clearly showed that Trichoderma viride was the significantly highest lipid producer, with lipid production at initial pH 6.0 and incubation temperature 40℃.