© 2019 Wastewater-based epidemiology is an increasingly popular method for analysing drugs or metabolites excreted by populations. The in-sewer transformation of biomarkers is important but often receives little consideration in published studies. Many studies publish stability under biofilm-free conditions only, which do not represent actual sewer conditions. This study aims to fill a gap in the field by comparing the wastewater stability of 33 licit drug and pharmaceutical biomarkers in biofilm-free (BFF) conditions to stability in sewer biofilm reactors. All but one biomarker was stable under BFF conditions, whereas most transformed in sewer biofilm reactors. Sewer reactor results tended to overestimate the degradation in pilot and actual sewers, whereas BFF stability had no clear relationship to stability in pilot and actual sewers. Our results provide additional basis for more informed interpretation of biofilm-free and sewer reactor stability results for past and future WBE studies.