Many organic contaminants entering the aquatic environment feature stereogenic structural elements that give rise to enantiomerism. While abiotic processes usually act identical on enantiomers, biotic processes, such as biodegradation often result in enantiomeric fractionation (EFr), i.e., the change of the relative abundance of enantiomers. Therefore, EFr offers the opportunity to differentiate biodegradation in complex environmental systems from abiotic processes. In this study, an achiral-chiral two-dimensional liquid chromatographic method for the enantioseparation of selected pharmaceuticals was developed. This method was then applied to determine the enantiomeric compositions of eight chiral pharmaceuticals in 20 water-sediment test flumes and test EFr as an indicator of biodegradation. While all eight substances were attenuated by at least 60%, five (atenolol, metoprolol, celiprolol, propranolol, and flecainide) displayed EFr. No EFr was observed for citalopram, fluoxetine, and venlafaxine despite almost complete attenuation (80 to 100%). Celiprolol, a barely studied β-blocker, revealed the most distinct EFr among all investigated substances; however, EFr varied considerably with biodiversity. Celiprolol-H2 was identified as a biological transformation product possibly formed by reduction of the celiprolol keto group through a highly regio- and enantioselective carbonyl reductase. While celiprolol-H2 was observed across all flumes, as expected, its formation was faster in flumes with high bacterial diversity where also EFr was highest. Overall, EFr and transformation product formation together served as good indicators of biological processes; however, the strong dependence of EFr on biodiversity limits its usefulness in complex environmental systems.