Increased seawater pCO₂ has the potential to alter phytoplankton biochemistry, which in turn may negatively affect the nutritional quality of phytoplankton as food for grazers. Our aim was to identify how Antarctic phytoplankton, Pyramimonas gelidicola, Phaeocystis antarctica, and Gymnodinium sp., respond to increased pCO₂. Cultures were maintained in a continuous culture setup to ensure stable CO₂ concentrations. Cells were subjected to a range of pCO₂ from ambient to 993 µatm. We measured phytoplankton response in terms of cell size, cellular carbohydrate content, and elemental, pigment and fatty acid composition and content. We observed few changes in phytoplankton biochemistry with increasing CO₂ concentration which were species-specific and predominantly included differences in the fatty acid composition. The C:N ratio was unaffected by CO₂ concentration in the three species, while carbohydrate content decreased in Pyramimonas gelidicola, but increased in Phaeocystis antarctica. We found a significant reduction in the content of nutritionally important polyunsaturated fatty acids in Pyramimonas gelidicola cultures under high CO₂ treatment, while cellular levels of the polyunsaturated fatty acid 20:5ω3, EPA, in Gymnodinium sp. increased. These changes in fatty acid profile could affect the nutritional quality of phytoplankton as food for grazers, however, further research is needed to identify the mechanisms for the observed species-specific changes and to improve our ability to extrapolate laboratory-based experiments on individual species to natural communities.