In many cases, only selected well-known target chemicals are analysed and used for a comparison with biological effects. Predicting the environmental impact of different chemical compounds does often fail. Effects are the result of mixture toxicity of single chemicals and their degraded products, which can have different biological potency and bioavailability. In vitro toxicity or mechanism-based assays are used as screening tools, prior to extended evaluation in animals or natural populations, or even prior to chemical monitoring. This study illustrates the use of the yeast bioassay to investigate the presence of compounds or chemicals with estrogenic activity in wastewater and surface water in Luxembourg and compares results with chemical measurements. Although the approach described in this paper has already been published in many case studies before, it confirms earlier findings and it delivers results for Luxembourg where similar analyses have not been documented so far. By comparison of the biological signal in the yeast assay, expressed as estrogen equivalents, with available results by liquid chromatography-mass spectrometry for steroid hormones we could only obtain a similar ranking for the majority of samples with low or high estrogenic activity. Measurements can therefore only be used as a screening tool for estrogenic activity. Seasonal changes as apparent for chemical results in surface waters were confirmed in the yeast assay. Dissolved estrone is diluted by higher discharge in the rivers during winter containing larger amounts of unpolluted soil water and groundwater runoff.