Attenuation of micro-contaminants is a very complex field in environmental science and evidence suggests that biodegradation rates of micro-contaminants in the aqueous environment depend on the water matrix. The focus of the study presented here is the systematic comparison of biotransformation rates of caffeine, carbamazepine, metoprolol, paracetamol and valsartan in river water microcosms spiked with different proportions of treated effluent (0%, 0.1%, 1%, and 10%). Biotransformation was identified as the dominating attenuation process by the evolution of biotransformation products such as atenolol acid and valsartan acid. Significantly decreasing biotransformation rates of metoprolol were observed at treated effluent proportions ≥0.1% whereas significantly increasing biotransformation rates of caffeine and valsartan were observed in the presence of 10% treated effluent. Potential reasons for the observations are discussed and the addition of adapted microorganisms via the treated effluent was suggested as the most probable reason. The impact of additional phosphorus on the biodegradation rates was tested and the experiments revealed that phosphorus-limitation was not responsible.