Caves are laboratories for many disciplines that work in natural sciences including mineralogy, biology, hydrogeology, and archaeology. In this study, bi-monthly samplings were carried out from three sampling locations within and around the Güvercinkaya Cave, a high-altitude cave located in northwestern Turkey, to evaluate the hydrochemical and microbiological properties and the aquatic macroinvertebrates of the cave stream. Some parameters of the water including pH, electrical conductivity, temperature, oxidation-reduction potential, and dissolved oxygen were measured in-situ, while elemental (70 in total) and ionic composition of water were analyzed in the laboratory. Microbiological analyses of the cave stream were examined through analyses of total bacteria, total coliforms, fecal coliforms, fecal Streptococcus, and Escherichia coli. According to the Piper diagram of hydrochemical data, the cave stream had mainly Ca-Mg-HCO3 character, on the other hand, the Schoeller diagram indicated a common water source in Güvercinkaya cave due to the similar components of the main ionic components of the water. As a result of microbiological analysis, fecal contamination was determined, indicating active wildlife in the cave. Additionally, several aquatic macroinvertebrates taxa, Rhynchelmis limosella, Dugesia sp., Gammarus uludagi which have non-troglobiont character were found in the cave stream. Rhynchelmis limosella detected in this study is the first record for the Turkish fauna.