Microalgae Isochrysis sp. and Myrmecia incisa have high nutritional value,which are rich in docosahexaenoic acid (DHA) and arachidonic acid (ArA) respectively. In the present study, the heterotrophic/mixotrophic strategies of these two microalgae were investigated. Results showed that when using glucose as the organic carbon source, both Isochrysis sp. and M. incisa were able to be heterotrophic, quickly accumulating biomass under complete darkness. The optimal glucose mass concentrations for Isochrysis sp. and M. incisa were 5 and 1 g/L, respectively. The effects of nitrogen mass in heterotrophic culture were also evaluated. For Isochrysis sp., 300 mg/L of sodium nitrate was the optimal for biomass production as well as for the intracellular accumulation of DHA. For M. incisa, nitrogen mass concentration had minor effect on its growth, but the impact on ArA production was profound, in which the deficiency of nitrogen induced the highest content of ArA. After demonstrating the heterotrophic properties of two algae and the optimal carbon and nitrogen supply level for them, the mixotrophic cultivation was further conducted, which led to significantly increased biomass and target polyunsaturated fatty acids. Finally, the algae obtained from mixotrophic condition were used to feed the Artemia sp.. Results showed that both DHA and ArA can be delivered from the microalgae to the Artemia sp., suggesting the potential as nutrient-enhanced live feed for aquaculture. Findings of the present study provided useful information for the exploitation and utilization of microalgal resources.