EPSs synthesized by thermophilic bacteria are natural biopolymers that have recently garnered attention due to their potential applications in areas such as pharmaceuticals and biomedicine. In this study, EPSs produced by five distinct thermophilic bacterial isolates from hot springs in Turkey were purified using ion exchange and gel chromatography, and the larvicidal and cytotoxic effects of these EPSs were examined. While Geobacillus thermodenitrificans HBB111 produces the highest quantity (650,9 µg/mL) of EPS, the protein content of crude EPS samples ranges from 0.3–1.5%. EPS111 and EPS261 showed the most effective larvicidal action, eliminating 72% and 62.7% of Ae. albopictus larvae after 48 hours, respectively. Among the purified samples, EPS111 exhibited the most significant effect on the proliferation of PC3 cells, resulting in a 68% inhibition (IC50 of 0,23 mg/mL) followed by EPS106 in a 55% (IC50 of 0,45 mg/mL). According to the results of our study, thermophilic EPSs demonstrate potential due to their insecticidal and anticancer properties.