The occurrence of pharmaceuticals in freshwater ecosystems provokes increasing concern due to their potential risk to non-target organisms and to human health. Pharmaceuticals are used in both human and veterinary medicine and are essentially released into the environment via wastewater treatment plants (WWTPs) and from livestock. In this study, 31 pharmaceuticals were analyzed in effluent and surface water upstream and downstream of two WWTPs in the Loue-Doubs rural karstic catchment in Eastern France. Diclofenac (965 and 2476 ng L-1), sulfamethoxazole (655 and 1380 ng L-1) and carbamazepine (566 and 1007 ng L-1) displayed the highest levels in the effluents of both WWTPs. Diclofenac levels were also high in surface water samples 300 and 166 ng L-1 in the River Doubs and the River Loue, respectively, followed by paracetamol (273 and 158 ng L-1) and sulfamethoxazole (126 and 73 ng L-1). In both rivers, the most critical compounds were found to be the antibiotic sulfamethoxazole (risk quotient (RQ) from 23.7 to 51.1) and ofloxacine (RQ from 1.1 to 18.9), which reached levels inducing toxic effects in aquatic organisms. This study showed that WWTP effluents are the major sources of the pharmaceuticals, but raw discharges from human residences, pastures and livestock manure represent significant sources of contamination of surface water and groundwater. The aim of this study was to assist scientists and authorities in understanding occurrence and sources of pharmaceuticals in order to improve water quality management in chalk streams.