The yeast estrogen screen was applied to sewage treatment process waters to identify the presence of estrogenic activity and to investigate the fate and behavior of estrogenic substances through treatment. Hydrophobic fractions in the water phase were extracted and concentrated using C18 cartridges for the effective extraction of 17β-estradiol (E2) and other estrogen mimics. Clear dose-dependent elevation in the synthesis of β-galactosidase in the yeast screen was observed with all the samples tested, demonstrating that these samples were estrogenic. However, estrogenic activity tended to reduce during the treatment, especiallyiin the biological process. Quantification results of the yeast estrogen screen in terms of E2 equivalent were compared with actual E2 concentrations measured by an ELISA. E2 occupied 34% of the whole estrogenicity in the raw sewage, while almost 100% in the final effluent. The analyses of the sewage treatment process waters revealed that human estrogens are major causative substances in terms of estrogenic activity in sewage and its treated effluent. Although findings of possible correlation of environmental estrogens with the real impact on human health and the ecosystem are still the focus of scientific debate and investigation, proper management should be established in the sewage treatment process which receives various environmental contaminants.