The geological formation of the Muschelkalk is widespread in the center of the North German Basin (NGB) and is increasingly attracting interest for application of geothermal energy extraction or high-temperature aquifer thermal energy storage (HT-ATES). This study investigates the Middle Triassic “Rüdersdorfer Schaumkalk”, which was the former injection horizon of the natural gas storage facility in Berlin, Germany. For the first time, detailed chemical and microbiological analyses of formation water of this Lower Muschelkalk limestone formation were conducted and hydrogeochemically characterized. In addition, a hydrogeochemical model was developed to quantify the potential reactions during HT-ATES focusing on calcite dissolution and precipitation. The main objectives of this study are: (1) to determine the origin of the water from the three wells targeting the Muschelkalk aquifer, (2) to understand changes in hydrochemistry after system operation, and (3) to evaluate the long-term sustainability of a potential HT-ATES system with increasing temperature. The target formation is encountered by several wells at about 525 m below the surface with an average thickness of 30 m. Two hydraulic lifting tests including physical, chemical, and microbial groundwater as well as gas monitoring were carried out. In addition, several downhole samples of formation fluid were collected from the aquifer at in situ pressure and temperature conditions. Fluid analysis of the saline formation water indicate a seawater origin within the Muschelkalk with subsequent evaporation and various water–rock interactions with anhydrite/gypsum, dolomite, and calcite. With a salinity of 130 g/L, dominated by Na–Cl, a slightly acidic pH between 6 and 7, and a low gas content of 3%, the formation water fits to other saline deep formation waters of the NGB. Gas concentrations and microbial communities like sulfate-reducing bacteria and methanogenic archaea in the produced water indicate several geochemical alterations and microbial processes like corrosion and the forming of biogenic methane. Geochemical simulations of calcite equilibrium over 10 HT-ATES cycles indicated a pronounced propensity for calcite precipitation up to 31 mg/kgw, within the heat exchanger. At the same time, these models predicted a significant potential for calcite dissolution, with rates up to 21 mg/kgw, in both the cold and hot reservoirs. The results from the carbonate aquifer characterized in this study can be transferred to other sites in the NGB affected by salt tectonics and have provided information on the microbiological-chemical processes to be expected during the initial use of old wells.