Mycotoxins are among the compounds of most concern for human health because of their common presence in food and their proven toxicity for human health. Human biomonitoring (HBM) studies, foodstuff analysis and dietary surveys are usually used to assess human exposure, but they are costly and time-consuming. Wastewater-based epidemiology (WBE) can complement the established exposure monitoring tools. The aim of this study was to develop a new WBE application for assessing human exposure to mycotoxins by measuring specific biomarkers in raw wastewater. Eleven substances were analysed by liquid chromatography-tandem mass spectrometry in wastewater from four cities in Italy and Spain. Only deoxynivalenol (DON) and fumonisines were found, DON being the most abundant, in accordance with HBM studies. DON intake, back-calculated by WBE, was lower than the established Health-Based Guidance values, indicating moderate exposure in the population, with no risks for human health. WBE intake estimates were very close to those reported in HBM studies, validating WBE for estimating DON intake in the population. This study describes a promising WBE approach to complement HBM studies and assess the human intake of specific classes of mycotoxins, thus helping to identify risks for human health.