Climate change has direct effects on aquatic systems where increased water temperature leads to range shifts and changes in the distribution of aquatic organisms. The effects of climate change on aquatic ecosystems are expected in all biomes, and in Alpine environments in particular. Anthropogenic pressure (e.g., chemical pollution) besides climate change impact on the state and quality of aquatic systems in which climate change and environmental contaminants can interact. To better understand the effect of increases in temperature and environmental pollution on high-mountain lakes, we performed an ecotoxicological assay on Daphnia middendorffiana collected during summer 2021 in a high-mountain lake (Upper Balma Lake, Cottian Alps, 2212 m a.s.L.). Samples were exposed to two temperature values (15 °C and 20 °C), potassium chloride (KCl) as the reference toxicant, and D. magna as the model organism for comparison. Findings showed immobilization after exposure to KCl in both species, but exposure to non-optimal water temperature (20 °C and 15 °C for D. middendorffiana and D. magna, respectively) enhanced this effect. The mean half-maximal effective concentration (EC50; 24 h) for D. middendorffiana was significantly lower than that recorded for D. magna exposed to 20 °C (KCl) (46.9 mg/L vs 255 mg/L). A significantly higher EC50 (273.4 mg/L; 24 h) was recorded for D. middendorffiana exposed to 15 °C (KCl) compared to D. magna (EC50 50.6 mg/L; 24 h). Our findings suggest that the combined effects of temperature and chemical pollution may severely affect the occurrence of D. middendorffiana, which occupies a central position in the food webs of high-mountain lakes in the Alps.