Despite evidence for microbial endemism, an understanding of the impact of geological and paleoclimate events on the evolution of regional protist communities remains elusive. Here, we provide insights into the biogeographical history of Antarctic freshwater diatoms, using lacustrine fossils from mid-Miocene and Quaternary Antarctica, and dovetail this dataset with a global inventory of modern freshwater diatom communities. We reveal the existence of a diverse mid-Miocene diatom flora bearing similarities with several former Gondwanan landmasses. Miocene cooling and Plio-Pleistocene glaciations triggered multiple extinction waves, resulting in the selective depauperation of this flora. Although extinction dominated, in situ speciation and new colonizations ultimately shaped the species-poor, yet highly adapted and largely endemic, modern Antarctic diatom flora. Our results provide a more holistic view on the scale of biodiversity turnover in Neogene and Pleistocene Antarctica than the fragmentary perspective offered by macrofossils and underscore the sensitivity of lacustrine microbiota to large-scale climate perturbations.