Phytoestrogens (isoflavones, enterolignans and coumestrol) in wastewater samples and surface water samples have been analysed by LC-ESI-MSⁿ. In wastewater samples, high levels of enterolactone (581–2111 ng/L), daidzein (341–1688 ng/L) and enterodiol (60–834 ng/L) were detected in raw sewage, but the vast majority of the analysed phytoestrogens were removed effectively in the treatment process. The removal rates of the analysed phytoestrogens in the two advanced tertiary treatment plants were >99%; a case study in one of the treatment plants showed that most of the residual phytoestrogens were removed by biological treatment using activated sludge. In surface water samples, daidzein was found at concentrations ranging from 2 ng/L to 33 ng/L in samples from two creeks, and up to 120 ng/L in surface water (pond) on a dairy farm. The analytical results suggest that direct excretions of livestock discharged from farmyards can be another potential source of phytoestrogen contamination in the aquatic environment.