This study was undertaken to investigate whether adaptation by pre-exposure to trace organic chemicals (TOrCs) was necessary for microbial transformation during managed aquifer recharge (MAR). Two pairs of laboratory-scale soil columns, each receiving a different primary substrate, were utilized to simulate the dominant bulk organic carbon present in MAR systems receiving wastewater effluent of varying quality and having undergone different degrees of pre-treatment, as well as organic carbon prevalent at different stages of subsurface travel. Each pair of columns consisted of duplicate set-ups receiving the same feed solution with only one pre-exposed to a suite of eight TOrCs for approximately ten months. Following the pre-exposure period, a spiking experiment was conducted in which the non-exposed columns also received the same suite of TOrCs. TOrC attenuation was quantified for the pre- and non-exposed columns of each pair during the spiking experiment. The microbial community structure and function of these systems were characterized by pyrosequencing of 16S rRNA gene and metagenomics, respectively. Biotransformation rather than sorption was identified as the dominant removal mechanism for almost all the TOrCs (except triclocarban). Similar removal efficiencies were observed between pre-exposed and non-exposed columns for most TOrCs. No obvious differences in microbial community structure were revealed between pre- and non-exposed columns. Using metagenomics, biotransformation capacity potentials of the microbial community present were also similar between pre- and non-exposed columns of each pair. Overall, the pre-exposure of MAR systems to TOrCs at ng/L levels did not affect their attenuation and had no obvious influence on the resulting microbial community structure and function. Thus, other factors such as bioavailability of the primary substrate play a greater role regarding biotransformation of TOrCs. These results indicate that MAR systems adapted to a primary substrate are capable of degrading TOrC without necessarily being pre-exposed to them, making MAR a robust treatment barrier for biodegradable TOrCs.