Aquaculture, the fastest growing food sector, is expected to expand to produce an additional 30 million metric tons of fish by 2030, thus filling the gap in supplies of seafood for humans. Salmonids aquaculture exploits the vast majority of fishmeal and fish oil rendered from ocean-dwelling forage fish. Most forage fish diverted to these commodities are human-food grade, and all are primary prey for marine predators. Rising costs, price volatility, and environmental sustainability concerns of using these commodities for aquaculture feed are driving the global search for alternatives, including marine microalgae originating from the base of marine food webs but produced in culture. We report the first evaluation of two marine microalgae, Nannochloropsis sp. and Isochrysis sp., for their potential to fully replace fishmeal and fish oil in diets of rainbow trout (Oncorhynchus mykiss), an important model for all salmonid aquaculture. We conducted a digestibility experiment with dried whole cells of Nannochloropsis sp. and Isochrysis sp., followed by a growth experiment using feeds with different combinations of Nannochloropsis sp., Isochrysis sp., and Schizochytrium sp. We found that digestibilities of crude protein, crude lipid, amino acids, fatty acids, omega 3 polyunsaturated fatty acids (n3 PUFA), docosahexaenoic acid (DHA), n6 (omega 6) PUFA in Isochrysis sp. were significantly higher than those in Nannochloropsis sp. Digestibility results suggest that for rainbow trout diets Isochrysis sp. is a better substitute for fishmeal and fish oil than Nannochloropsis sp. The lower feed intake by fish fed diets combining multiple microalgae, compared to fish fed the reference diet, was a primary cause of the growth retardation. In trout fillets, we detected an equal amount of DHA in fish fed fish-free diet and reference diet. This study suggests that Isochrysis sp. and Schizochytrium sp. are good candidates for DHA supplementation in trout diet formulations.