Background and Objective: Endocrine disruptors, emerging contaminants of concern, are mainly spread in the environment through effluents of wastewater treatment plants (WWTPs), so an efficient monitoring of effluents is needed. Biological monitoring through estrogenic activity assays has been suggested to improve water quality assessment. This monitoring can evaluate the overall endocrine activity, considering all chemicals with the same mode of action. However, it could be influenced by many factors. This study compared the estrogenic activity of wastewaters extracted with different extraction phases and analyzed with different assays to establish whether phase and/or assay may affect monitoring results. Methods: The effluents of six Italian WWTPs were collected in four seasonal samplings and extracted using two phases (HLB, C18). The extracts were tested using gene reporter assay on mammalian cells and yeast estrogen screen assay (YES). To assess whether the estrogenic activity posed an acceptable risk, Results of the cumulative effluent (discharged in surface waters) were compared with safe estrogenic levels reported in literature. Results: The estrogenic activity of effluents extracted with HLB was significantly different from the estrogenic activity of effluents extracted with C18, showing that phase type can affect monitoring results. Moreover, the Results were also affected by the assay type. Using the gene reporter assay the estrogenic activity was generally higher than using YES, confirming that mammalian cell-based assays are more sensitive than yeast-based assays. Finally, gene reporter data suggested that estrogenic activity of the cumulative effluent might pose a risk to the receiving waters, while YES data suggested that it does not represent an environmental threat. Conclusions: This study highlighted the need to define a standardized approach to assess the endocrine disrupting potential of waters and provided data that could be useful to choose the most appropriate extraction phase and assay to safeguard environment, thus protecting human health.