Estrogenic activity has been detected in aquatic ecosystems across the world. However, there is a lack of such data for Slovenian wastewaters and surface waters. The Slovenian monitoring program of effluents discharged into surface waters does not require that emissions of natural and synthetic estrogens into aquatic environments be assessed and controlled. In our study, we assessed the potential estrogenicity of wastewater samples from three wastewater treatment plants using a yeast estrogen screen assay (YES assay). Due to the high inhibition of yeast growth in samples obtained during our first sampling period, it was impossible to detect any estrogenic activity. An additional silica gel clean-up step reduced the toxicity of samples collected during our second sampling period; as a result, we were able to record up to 95% relative estrogenic activity inhibition. Deconjugation of the estrogens did not significantly influence our results. We detected estrogenic activity using a YES assay in almost all influent and effluent samples tested, which suggests that the wastewater treatment plants (WWTPs) examined do not effectively remove (xeno)estrogens from wastewaters. Our results suggest that a YES assay is an appropriate screening method for monitoring estrogenic activity in effluents. However, prediction of the potential impacts of wastewater (xeno)estrogens on aquatic organisms require additional in vitro and in vivo assays.