Only recently, a novel anoxic hypersaline (thalassic) basin in the eastern Mediterranean was discovered at a depth of 3,258 m. The halite-saturated brine of this polyextreme basin revealed one of the highest salt concentrations ever reported for such an environment (salinity of 348‰). Using a eukaryote-specific probe and fluorescence in situ hybridization, we counted 0.6 × 10(4) protists per liter of anoxic brine. SSU rRNA sequence analyses, based on amplification of environmental cDNA identified fungi as the most diverse taxonomic group of eukaryotes in the brine, making deep-sea brines sources of unknown fungal diversity and hotspots for the discovery of novel metabolic pathways and for secondary metabolites. The second most diverse phylotypes are ciliates and stramenopiles (each 20%). The occurrence of closely related ciliate sequences exclusively in other Mediterranean brine basins suggests specific adaptations of the respective organisms to such habitats. Betadiversity-analyses confirm that microeukaryote communities in the brine and the interface are notably different. Several distinct morphotypes in brine samples suggest that the rRNA sequences detected in Thetis brine can be linked to indigenous polyextremophile protists. This contradicts previous assumptions that such extremely high salt concentrations are anathema to eukaryotic life. The upper salinity limits for eukaryotic life remain unidentified.