Organic micropollutants of anthropogenic origin in river waters may impair aquatic ecosystem health and drinking water quality. To evaluate micropollutant fate and turnover on a catchment scale, information on input source characteristics as well as spatial and temporal variability is required. The influence of tributaries from agricultural and urban areas and the input of wastewater were investigated by grab and Lagrangian sampling under base flow conditions within a 7.7‐km‐long stretch of the Ammer River (southwest Germany) using target screening for 83 organic micropollutants and 4 in vitro bioassays with environmentally relevant modes of action. In total, 9 pesticides and transformation products, 13 pharmaceuticals, and 6 industrial and household chemicals were detected. Further, aryl hydrocarbon receptor induction, peroxisome proliferator–activated receptor activity, estrogenicity, and oxidative stress response were measured in the river. The vast majority of the compounds and mixture effects were introduced by the effluent of a wastewater‐treatment plant, which contributed 50% of the total flow rate of the river on the sampling day. The tributaries contributed little to the overall load of organic micropollutants and mixture effects because of their relatively low discharge but showed a different chemical and toxicological pattern from the Ammer River, though a comparison to effect‐based trigger values pointed toward unacceptable surface water quality in the main stem and in some of the tributaries. Chemical analysis and in vitro bioassays covered different windows of analyte properties but reflected the same picture. Environ Toxicol Chem 2020;39:1382–1391. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.