Pester Plateau is a unique karst region in Serbia, notable for its ephemeral and permanent springs. Its rich biodiversity, combined with distinctive geomorphological, geological, hydrological, and climatic features, shapes the fauna of benthic organisms inhabiting these springs. The porous soil and low filtration capacity make the springs highly vulnerable to pollution due to strong underground connections. A two-year study analyzed physical and chemical parameters, assessed water quality using macrozoobenthos, examined faunal diversity and community structure, identified new taxa, and proposed conservation measures and reference site designation for the karst springs of Pester Plateau, highlighting macrozoobenthos as a mirror of karst ecosystems. The dominant species across samples was Gammarus balcanicus (Schaferna 1922). The most diverse macroinvertebrate group included aquatic insects, such as Trichoptera (45 species), Plecoptera (19), Ephemeroptera (14), and Coleoptera (9 taxa). The rare circumpolar species Arcynopteryx dichroa (McLachlan 1872) was also recorded. Several species listed as strictly protected (“Official Gazette of RS,” No. 5/2010) and endangered in Serbia were found, including Dinocras megacephala, Nemoura cambrica, Protonemura meyeri, Protonemura praecox, Baetis pavidus, Allogamus auricollis, and Thremma anomalum. To assess ecological status and environmental pressures, macrozoobenthos communities were evaluated using the saprobic index, diversity index, BMWP score, and EPT index.