The fate of pharmaceutically active substances in rivers is still only incompletely understood, especially as the knowledge transfer from laboratory experiments to the real world is complicated by factors like turbidity, hydrodynamics, or heterogeneity. Therefore, we performed a tracer test with pharmaceutically active substances to study their fate and the importance of individual attenuation mechanisms in situ. The experiment was carried out at a small stream in central Sweden. Two dye tracers and six pharmaceuticals were injected as Dirac pulse and water was sampled at five downstream sites along a 16-km-long river reach. Ibuprofen and clofibric acid were the only compounds which were eliminated along the study reach at half-life times of 10 h and 2.5 d, respectively. Based on the shape of the breakthrough curves and the low hydraulic conductivity of the river bed, we can assume that exchange of river water with the hyporheic zone was minor. Thus, the contribution of processes in the hyporheic zone to the attenuation of pharmaceuticals was low. We hypothesize that ibuprofen and clofibric acid were transformed by in-stream biofilms growing on submerged macrophytes and at the water-sediment interface. Phototransformation and sorption were ruled out as major attenuation processes. No attenuation of bezafibrate, diclofenac, metoprolol, and naproxen was observed.