The effects of “trace” (environmentally relevant) concentrations of the antimicrobial agent sulfamethoxazole (SMX) on the growth, nitrate reduction activity, and bacterial composition of an enrichment culture prepared with groundwater from a pristine zone of a sandy drinking-water aquifer on Cape Cod, MA, were assessed by laboratory incubations. When the enrichments were grown under heterotrophic denitrifying conditions and exposed to SMX, noticeable differences from the control (no SMX) were observed. Exposure to SMX in concentrations as low as 0.005 μM delayed the initiation of cell growth by up to 1 day and decreased nitrate reduction potential (total amount of nitrate reduced after 19 days) by 47% (p = 0.02). Exposure to 1 μM SMX, a concentration below those prescribed for clinical applications but higher than concentrations typically detected in aqueous environments, resulted in additional inhibitions: reduced growth rates (p = 5 × 10⁻⁶), lower nitrate reduction rate potentials (p = 0.01), and decreased overall representation of 16S rRNA gene sequences belonging to the genus Pseudomonas. The reduced abundance of Pseudomonas sequences in the libraries was replaced by sequences representing the genus Variovorax. Results of these growth and nitrate reduction experiments collectively suggest that subtherapeutic concentrations of SMX altered the composition of the enriched nitrate-reducing microcosms and inhibited nitrate reduction capabilities.