A complex mixture of emerging contaminants (ECs) occurs in the environment, with potential effects for aquatic organisms and human health. This study assessed the environmental risk of a mixture of ECs detected in the most urbanized and industrialized area of Italy. Water samples were collected in the Lambro river basin to the north and south of Milan, and were analyzed by liquid chromatography tandem mass spectrometry. The Environmental Risk Assessment (ERA) was performed calculating the Risk Quotients (RQs) for each EC as the ratio of Measured Concentrations (MECs) and Predicted No Effect Concentrations. The ERA was also conducted for the whole mixture of ECs by considering the RQs of the components. The results confirmed that ECs are ubiquitous in urbanized areas. The ERA for the single pollutants identified a panel of substances of environmental concern (estrogens, amoxicillin, clarythromycin, triclosan and nicotine). The ERA for the mixture indicated a potential cumulative risk for the substances that individually could be considered safe, highlighting the importance of taking the whole mixture of ECs into account for the ERA. This information may help establish EU regulations for ECs and environmental quality standards for regulatory purposes.