Pharmaceuticals find their way to the aquatic environment via wastewater treatment plants (WWTPs). Biotransformation plays an important role in mitigating environmental risks; however, a mechanistic understanding of involved processes is limited. The aim of this study was to evaluate potential relationships between first-order biotransformation rate constants (k(b)) of nine pharmaceuticals and initial concentration of the selected compounds, and sampling season of the used activated sludge inocula. Four-day bottle experiments were performed with activated sludge from WWTP Groesbeek (The Netherlands) of two different seasons, summer and winter, spiked with two environmentally relevant concentrations (3 and 30 nM) of pharmaceuticals. Concentrations of the compounds were measured by LC-MS/MS, microbial community composition was assessed by 16S rRNA gene amplicon sequencing, and k(b) values were calculated. The biodegradable pharmaceuticals were acetaminophen, metformin, metoprolol, terbutaline, and phenazone (ranked from high to low biotransformation rates). Carbamazepine, diatrizoic acid, diclofenac, and fluoxetine were not converted. Summer and winter inocula did not show significant differences in microbial community composition, but resulted in a slightly different k(b) for some pharmaceuticals. Likely microbial activity was responsible instead of community composition. In the same inoculum, different k(b) values were measured, depending on initial concentration. In general, biodegradable compounds had a higher k(b) when the initial concentration was higher. This demonstrates that Michealis-Menten kinetic theory has shortcomings for some pharmaceuticals at low, environmentally relevant concentrations and that the pharmaceutical concentration should be taken into account when measuring the k(b) in order to reliably predict the fate of pharmaceuticals in the WWTP.