OBJECTIVES: The purpose of this study was to determine toxicity of wastewater from hospitals in the Czech Republic using traditional and alternative toxicological methods. The pilot study comprised weekly dynamics of sewage ecotoxicity of treated wastewater from one hospital in two different seasons. A detailed investigation of wastewater ecotoxicity, genotoxicity and reprotoxicity followed in five different hospitals. METHODS: The seven following bioassays were used in this study: algal growth inhibition test (ISO 8692), Vibrio fischeri test (ISO 11348-2), Daphnia magna acute toxicity test (ISO 6341), Allium cepa assay, Ames test (OECD TG 471), Comet assay and YES/YAS assay. RESULTS: The wastewater ecotoxicity during one week showed no differences in separate working days, however, higher toxicity values were recorded in May compared to November. In the following study, samples from two of the five hospitals were classified as toxic, the others as non toxic. Genotoxicity has not been confirmed in any sample. In several cases, wastewater samples exhibited agonist activity to the estrogen and androgen receptors. CONCLUSION: The study demonstrated different levels of toxicity of treated hospital wastewater. Variable sensitivity of individual bioassays for tested wastewater samples was recognized. A more extensive study including proposal for improvement of hospital wastewater treatment within the Czech Republic can be recommended with the aim to decrease the discharge of toxic chemicals into the local sewage system and the environment.