The capacity of rivers for biological attenuation processes of trace polar organic pollutants are probably significant but poorly understood because the many factors (i.e., dilution, sorption, phototransformation, biotransformation) that control naturally attenuation are interrelated and difficult to study in isolation. Although it is possible to estimate in-stream attenuation rates from monitoring data, accurate estimates require large data sets. To overcome these limitations, chirality has been exploited in this work since only biological processes might change the enantiomer composition of chemicals. The main aim of this work was to investigate the chiral signature of the antidepressant venlafaxine and the β-blocker metoprolol to get insights into biological attenuation processes in river environment. This work includes laboratory-scale experiments to assess the contribution of sorption, phototransformation and biotransformation processes to the overall attenuation of venlafaxine and metoprolol as well as field observations to validate laboratory results. During base flow conditions (summer), enantiomeric fractions (EF) of each compound remained constant while they fluctuated during rainfall events (fall). High flow conditions probably prompted the release of chemicals accumulated during the dry season in sediment, where enantioselective biotransformation probably took place. Investigating chiral signature of selected pharmaceuticals is a promising tool not only to discriminate between abiotic and biotic transformation processes in river but also to assess the spatial and temporal variability in biologicalattenuation processes.