AbstractKarst caves, formed from the dissolution of soluble rocks, are characterized by the absence of photosynthetic activity and low levels of organic matter. Organisms evolve under these particular conditions, which causes high levels of endemic biodiversity in both macroorganism and microbes. Recent research has highlighted the presence of testate amoebae (Arcellinida) group in cave environments. This study investigates the diversity of Arcellinida in Dinaric karstic caves in Croatia, a global diversity hotspot, focusing on the influence of bat guano on community structure. Sediment samples were collected from two independent hydrosystems, and a metabarcoding approach was used to assess Arcellinida diversity at specific and intraspecific levels, using Arcellinid-specific primers to amplify the mitochondrial cytochrome oxidase subunit I (COI) region. Results reveal a significant impact of guano on both specific and intraspecific diversity of Arcellinida. Communities in guano-rich sites displayed higher diversity, abundance, and the presence of unique OTUs and genetic variants not observed in other habitats, highlighting the crucial role of bats as ecosystem engineers. In contrast, sites without guano hosted communities with low abundance and reduced biodiversity. These differences suggest the existence of guano-associated Arcellinida communities. This study provides new insights into the biodiversity of subterranean ecosystems and the ecological roles of Arcellinida in karstic environments.