Many studies demonstrated the presence of diverse environmental contaminants in the Douro River estuary, such as natural and synthetic estrogens, pharmaceuticals, industrial compounds and pesticides. This estuary is located between two densely populated cities and is highly impacted due to anthropogenic activities, such as industry and agriculture. Although the presence of mycotoxins and phytoestrogens, such as lignans and coumestrans, in the aquatic environment is reported by some authors, their occurrence in Portuguese waters was not investigated yet. To evaluate the presence of phytoestrogens, phytosterols and mycotoxins in Douro River estuary, water samples were collected seasonally at nine sampling points, preconcentrated by solid phase extraction and analysed by gas chromatography mass spectrometry. Local flora was collected on the riverside, in the same sampling points, for identification and evaluation of the possible relation to the presence of phytoestrogens and/or phytosterols in the estuarine water. Results showed the ubiquitous presence of mycotoxins, namely deoxynivalenol up to 373.5 ng L(-1). Both phytoestrogens and phytosterols showed a possible seasonal fluctuation, which is in accordance to the life cycle of the local flora and agricultural practices. Physicochemical parameters were also determined for water quality evaluation. This study revealed for the first time the presence of mycotoxins and lignans in estuarine waters from Portugal, and highlights the need to consider natural contaminants in future monitoring programs.