Croatian geothermal waters represent poorly explored locations primarily used for balneological purposes. The microbiological aspects of these waters in the context of biotechnological potential have not been studied. In the first and second research phases, the prokaryotic and eukaryotic microbial communities were analyzed in 19 (prokaryotes) and 14 (eukaryotes) different geothermal locations, by 16S and 18S rRNA sequencing. The results revealed significant diversity among thermophilic prokaryotes, with the most abundant representatives being Proteobacteria, Campylobacteria, and Crenarchaeota. The communities were often dominated by a single genus (Hydrogenophilus, Sulfuricurvum, Sulfurovum, Thiofaba, Nitrospira, Ca. Nitrosotenuis, and Ca. Nitrososphaera). No seasonal variability was observed. Among the microbial eukaryotes in autumn 2020., representatives of various functional groups were found, including primary producers and decomposers (Tribonema, Navicula, Cymbella, Spirogyra, Chromulina, Fungi, Sagenista). Statistical analyses in both research phases indicated the importance of abiotic factors such as temperature, pH value, and nutrients in microbial community shaping. Based on the obtained results and the dominance of prokaryotic communities associated with nitrogen (AOA, NOB, comammox) and sulfur cycles (SOB, SRB), Croatian geothermal waters have the potential to produce a wide range of bacterial and archaeal enzymes that can be applied in bioremediation. The results regarding microbial eukaryotes in Croatian geothermal waters highlight the ecological significance of microalgae and their potential use in biogas production, and bioremediation. The third research phase involved the cultivation of thermophilic prokaryotes involved in the nitrification process. The cultures' most common genera associated with the nitrogen cycle were Meiothermus, Nitrospira, Candidatus Nitrososphaera, Ca. Nitrosotenuis, and Denitratisoma. Some cultures were used in the biological treatment of biowaste leachate in batch systems at 25 °C, where they additionally reduced the total organic load and contributed to the improvement of the bioremediation process. The comprehensive investigation of microbial community composition and diversity in extreme habitats is crucial for future research of their metabolic processes at geothermal sites, which exhibit significant potential for biotechnological applications.