Abstract The enantiomeric and diastereomeric profiling of chiral pharmaceuticals (ephedrine, norephedrine, atenolol and venlafaxine) and illicit drugs (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-methylamphetamine (MDMA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA)) was undertaken over a period of fourteen consecutive days in three wastewater treatment plants (WWTPs) in the city of Valencia, Spain. Degradation efficiency of WWTPs was found to be compound and enantiomer dependent. Selective enantiomer enrichment was observed for several target analytes. Amphetamine and MDMA were enriched with R(−)-enantiomers. 1S,2S(+)-pseudoephedrine was found to be more readily degradable during activated sludge treatment than its diastereomer 1R,2S(−)-ephedrine. Atenolol underwent enrichment with either S(−)- or R(+)-enantiomer in different WWTPs. This unexpected enantiomeric variation in the stereoselective degradation of atenolol could be attributed to different processes utilized during activated sludge treatment. The application of (enantiomeric) profiling of wastewater revealed usage patterns of chiral drugs in the Valencia region.