There is increasing awareness that chemical pollution of freshwater systems with complex mixtures of chemicals from domestic sources, agriculture and industry may cause a substantial chemical footprint on water organisms, pushing aquatic ecosystems outside the safe operating space. The present study defines chemical footprints as the risk that chemicals or chemical mixtures will have adverse effects on a specific group of organisms. The aim is to characterise these chemical footprints in European streams based on a unique and uniform screening of more than 600 chemicals in 445 surface water samples, and to derive site- and compound-specific information for management prioritisation purposes. In total, 504 pesticides, biocides, pharmaceuticals and other compounds have been detected, including frequently occurring and site-specific compounds with concentrations up to 74 µg/L. Key finding is that three-quarter of the investigated sites in 22 European river basins exceed established thresholds for chemical footprints in freshwater, leading to expected acute or chronic impacts on aquatic organisms. The largest footprints were recorded on invertebrates, followed by algae and fish. More than 70 chemicals exceed thresholds of chronic impacts on invertebrates. For all organism groups, pesticides and biocides were the main drivers of chemical footprints, while mixture impacts were particularly relevant for invertebrates. No clear significant correlation was found between chemical footprints and the urban discharge fractions, suggesting that effluent-specific quality rather than the total load of treated wastewater in the aquatic environment and the contribution of diffuse sources, e.g. from agriculture, determine chemical footprints.