Recent studies have clearly shown the importance of omega-3 (ω-3) and omega-6 (ω-6) polyunsaturated fatty acids (PUFAs) for human and animal health. The long-chain eicosapentaenoic acid (EPA; 20:5ω-3) and docosahexaenoic acid (DHA; 22:6ω-3) are especially recognized for their nutritional value, and ability to alleviate many diseases in humans. So far, fish oil has been the main human source of EPA and DHA, but alternative sources are needed to satisfy the growing need for them. Therefore, we compared a fatty acid profile and content of 10 diatoms and seven dinoflagellates originating from marine, brackish and freshwater habitats. These two phytoplankton groups were chosen since they are excellent producers of EPA and DHA in aquatic food webs. Multivariate analysis revealed that, whereas the phytoplankton group (46%) explained most of the differences in the fatty acid profiles, habitat (31%) together with phytoplankton group (24%) explained differences in the fatty acid contents. In both diatoms and dinoflagellates, the total fatty acid concentrations and the ω-3 and ω-6 PUFAs were markedly higher in freshwater than in brackish or marine strains. Our results show that, even though the fatty acid profiles are genetically ordered, the fatty acid contents may vary greatly by habitat and affect the ω-3 and ω-6 availability in food webs.