The Dinarides of Bosnia and Herzegovina are exceptionally rich in surface and subterranean geomorphological and hydrological karst features which make them an ideal setting for ecological studies of microorganisms. This work presents the results of a seven-year study of testate amoebae, polyphyletic eukaryotic microorganisms, across various karst habitats. The main objectives were to collect data on testate amoebae biodiversity and to deepen our understanding of the transformation of their assemblages across surface and subterranean habitats. More than 50 taxa of testate amoebae were identified, including the notable finding of Psammonobiotus dinarica – a species originally described from caves – in a pristine surface karst river. A Jaccard resemblance-based cluster analysis of overall testate amoebae biodiversity revealed a clear separation of lake assemblages from river, spring, soil and cave assemblages. These results were confirmed by site-based cluster analysis and NMDS ordination, which showed strong separation (SIMPROF test) of lake, soil and cave assemblages, but also distinct assemblages of hydrologically interconnected karst features (sinking river Šuica → sinkhole cave Ponor Kovači → the resurgence Vrilo Spring). This study confirms a high similarity between river, spring, cave, and soil assemblages but reveals a previously unknown distinction of lake assemblages. It also showed that spring assemblages have higher similarity to river assemblages than to those in caves, implying a possible impact of primary production due to diffuse light in springs during summer droughts. This study emphasizes the need for further research of microbial assemblages in relation to environmental factors in order to support science-based protection of both surface and subterranean karst ecosystems.