This paper describes the development and application of a multi-residue chiral liquid chromatography coupled with tandem mass spectrometry method for simultaneous enantiomeric profiling of 18 chiral pharmaceuticals and their active metabolites (belonging to several therapeutic classes including analgesics, psychiatric drugs, antibiotics, cardiovascular drugs and β-agonists) in surface water and wastewater. To the authors' knowledge, this is the first time an enantiomeric method including such a high number of pharmaceuticals and their metabolites has been reported. Some of the pharmaceuticals have never been studied before in environmental matrices. Among them are timolol, betaxolol, carazolol and clenbuterol. A monitoring programme of the Guadalquivir River basin (South Spain), including 24 sampling sites and five wastewater treatment plants along the basin, revealed that enantiomeric composition of studied pharmaceuticals is dependent on compound and sampling site. Several compounds such as ibuprofen, atenolol, sotalol and metoprolol were frequently found as racemic mixtures. On the other hand, fluoxetine, propranolol and albuterol were found to be enriched with one enantiomer. Such an outcome might be of significant environmental relevance as two enantiomers of the same chiral compound might reveal different ecotoxicity. For example, propranolol was enriched with S(-)-enantiomer, which is known to be more toxic to Pimephales promelas than R(+)-propranolol. Fluoxetine was found to be enriched with S(+)-enantiomer, which is more toxic to P. promelas than R(-)-fluoxetine.