Pollution of drinking water sources represents a continuously emerging problem in global environmental protection. Novel techniques for real-time monitoring of water quality, capable of the detection of unanticipated toxic and bioactive substances, are urgently needed. In this study, the applicability of a cell-based sensor system using selected eukaryotic cell lines for the detection of aquatic pollutants is shown. Readout parameters of the cells were the acidification (metabolism), oxygen consumption (respiration) and impedance (morphology) of the cells. A variety of potential cytotoxic classes of substances (heavy metals, pharmaceuticals, neurotoxins, waste water) was tested with monolayers of L6 cells (rat myoblasts). The cytotoxicity or cellular effects induced by inorganic ions (Ni(2+) and Cu(2+)) can be detected with the metabolic parameters acidification and respiration down to 0.5 mg/L, whereas the detection limit for other substances like nicotine and acetaminophen are rather high, in the range of 0.1 mg/L and 100 mg/L. In a close to application model a real waste water sample shows detectable signals, indicating the existence of cytotoxic substances. The results support the paradigm change from single substance detection to the monitoring of overall toxicity.