Pester Plateau is an exceptionally important region in Serbia, standing out as one of the last large and still preserved upland karst fields. Its most prominent feature is the presence of ephemeral and permanent waters. In addition to its rich biodiversity, Pester Plateau is characterized by unique geomorphological, eological, hydrogeological, hydrological, and climatic phenomena, as well as a distinctive landscape physiognomy. These factors, through their combined influence, shape the fauna of the benthic organisms in the ephemeral and permanent waters of Pester Plateau. Due to the porous nature of the soil in this area, the filtration efficiency is low, making these areas highly susceptible to pollution, especially since the underground parts are well-connected to surface springs. The conducted study, spanning two years, aimed to analyze the physical and chemical parameters of ephemeral and permanent waters on Pester Plateau. The water quality of the examined springs and streams on Pester Plateau was assessed using macrozoobenthos, along with the analysis of metric characteristics of the communities. The research focused on exploring the faunal composition and diversity of macrozoobenthos in the examined springs and streams of Pester Plateau, with a particular emphasis on discovering new taxa. The determination of abiotic and biotic factors influencing the diversity of macroinvertebrates in the permanent and ephemeral karst springs and streams of Pester Plateau was also undertaken. Proposed measures for the protection and conservation of aquatic ecosystems on Pester Plateau, as well as suggestions for categorization and formation of reference sites for the examined springs and streams, were provided. During this study, 163 taxa were identified through quantitative sampling, of which 132 were determined at the species level, 29 at the genus level, while only two taxa were identified at higher taxonomic categories. The dominant group in the complete sample was Gammaridae, representing 40.17% with one species, Gammarus balcanicus (Schaferna, 1922). However, the most diverse group of macroinvertebrate communities consisted of aquatic insects, including Trichoptera (68 species), Ephemeroptera (26), Plecoptera (21), and Coleoptera (19 taxa). The study also identified the species Metreletus balcanicus (Ulmer, 1920), which represents a new genus for Serbia, and the species Arcynopteryx dichroa (McLachlan, 1872), a circumpolar and rare species in Serbia. Several species listed as strictly protected species (“Official Gazette of RS,” No. 5/2010) and endangered for the territory of Serbia were observed, including Dinocras megacephala (Klapalek, 1907), Nemoura cambrica (Stephens, 1836), Protonemura meyeri (Pictet, 1841), Protonemura praecox (Morton, 1894), Baetis pavidus (Grandi, 1951), Allogamus auricollis (Pictet, 1834), and Thremma anomalum (McLachlan, 1876). Analysis of variance revealed a statistically significant difference in the macrozoobenthos community between Pester Field and Koštanpolje on Pester Plateau, but not between the localities on Jare and the outskirts of Pester Plateau, which was confirmed by cluster analysis, distinguishing two clusters, one in Pester Field and the other in Koštanpolje.