This is the first report on the diversity of small free-living amoebae (FLA) in carbonate precipitating habitats in karst caves. Of 11 samples from nine different habitats in four Slovenian karst caves ten samples were positive for FLA, four strains were successfully isolated and transferred to clonal monoxenic cultures, including Acanthamoeba castellanii genotype T4, Echinamoeba silvestris, Hartmannella vermiformis, and a new vahlkampfiid amoeba Allovahlkampfia spelaea gen. nov., sp. nov. The latter was isolated from a stromatolitic stalagmite, a typical biogenic speleothem. Echinamoeba silvestris was identified from an aerophytic algal community and Acanthamoeba and Hartmannella were isolated from a cave pool with floating calcite rafts. The grazing of FLA on bacteria may help in creating conditions that enhance carbonate precipitation.