Trace organic contaminants (TrOCs) are omnipresent in wastewater treatment plants (WWTPs), yet, their removal during wastewater treatment is oftentimes incomplete and underlying biotransformation mechanisms are not fully understood. In this study, we elucidate how different factors, including pre-exposure levels and duration, influence microbial adaptation towards catabolic TrOC biodegradation and its potential role in biological wastewater treatment. Four sequencing batch reactors (SBRs) were operated in parallel in three succeeding phases, adding and removing a selection of 26 TrOCs at different concentration levels. After each phase of SBR operation, a series of batch experiments was conducted to monitor biotransformation kinetics of those same TrOCs across various spike concentrations. For half of our test TrOCs, we detected increased biotransformation in sludge pre-exposed to TrOC concentrations ≥5 µg L⁻¹ over a 30-day period, with most significant differences observed for the insect repellent DEET and the artificial sweetener saccharin. Accordingly, 16S rRNA amplicon sequencing revealed enrichment of taxa that have previously been linked to catabolic biodegradation of several test TrOCs, e.g., Bosea sp. and Shinella sp. for acesulfame degradation, and Pseudomonas sp. for caffeine, cyclamate, DEET, metformin, paracetamol, and isoproturon degradation. We further conducted shotgun metagenomics to query for gene products previously reported to be involved in the TrOCs’ biodegradation pathways. In the future, directed microbial adaptation may be a solution to improve bioremediation of TrOCs in contaminated environments or in WWTPs.