Diatoms of the genus Pseudo-nitzschia, known for their potential toxicity, are integral to the phytoplankton community of the Southern Ocean, which surrounds Antarctica. Despite their ecological importance, the diversity and toxicity of Pseudo-nitzschia in this region remain underexplored. Globally, these diatoms are notorious for forming harmful algal blooms in temperate and tropical waters, causing significant impacts on marine life, ecosystems, and coastal economies. However, detailed information on the diversity, morphology, and toxicity of Pseudo-nitzschia species in Antarctic waters is limited, with molecular characterizations of these species being particularly scarce. During three research expeditions to the Southern Ocean, monoclonal strains of Pseudo-nitzschia were isolated and cultivated. Stored samples from a fourth expedition, the Brategg expedition, were used to complete the description of particularly P. turgidula. Through electron microscopy and molecular analysis, two novel species were identified—Pseudo-nitzschia meridionalis sp. nov. and Pseudo-nitzschia glacialis sp. nov.—alongside the previously described species P. subcurvata, P. turgiduloides, and P. turgidula. Toxin assays revealed no detectable levels of domoic acid in P. turgiduloides, P. turgidula, P. meridionalis sp. nov. and P. glacialis sp. nov. Conversely, P. subcurvata was reported in a related study to produce domoic acid and its isomer, isodomoic acid C. These findings emphasize the need for comprehensive research on the phytoplankton of Antarctic waters, which is currently a largely uncharted domain. With the looming threat of climate change, understanding the dynamics of potentially harmful algal populations in this region is becoming increasingly critical.