Dinoflagellates contain large amounts of omega-3 fatty acids, including the nutritionally important docosahexaenoic acid (DHA). However, their cultivation in suspensions is characterized by low growth rates. Twin-layer porous substrate photobioreactors (TL-PSBRs) have been shown to support growth of different microalgal species, including the robust dinoflagellate Symbiodinium voratum. In the present study, the potential of cultivating marine autotrophic dinoflagellate species in a TL-PSBR for DHA production was explored. Based on initial screening experiments, two Symbiodinium species with high biomass and DHA productivities were selected: the symbiotic Symbiodinium microadriaticum CCAC 2475 B and the free-living Symbiodinium voratum CCAC 3869 B. The effects of three different temperatures (17, 22 and 27 °C) and nitrogen regimes (nitrate, ammonium and nitrogen-free) on biomass growth, total lipid accumulation and fatty acid methyl esters (FAMEs) content, with emphasis on DHA, were evaluated. The two lower temperatures (17 and 22 °C) enhanced growth and total lipid accumulation of S. microadriaticum CCAC 2475 B and S. voratum CCAC 3869 B. Cultivation at 22 °C and nitrogen limitation led to a significant positive effect on DHA productivity. Symbiodinium. microadriaticum CCAC 2475 B reached a DHA productivity of 145.4 mg m−2 day−1 and DHA content in the dry biomass of 2% (w/w) after 4 days of nitrogen depletion. The results of the present study demonstrated that autotrophic dinoflagellates, when cultivated on a TL-PSBR, produce comparable amounts of lipids and fatty acids to other commercially used microalgal species including the valuable DHA.