Effects upon microbial communities from environmental exposure to concentrations of antibiotics in the μg L^–1 range remain poorly understood. Microbial communities from an oligotrophic aquifer (estimated doubling rates of only once per week) that were previously acclimated (AC) or unacclimated (UAC) to historical sulfamethoxazole (SMX) contamination, and a laboratory-grown Pseudomonas stutzeri strain, were exposed to 240–520 μg L^–1 SMX for 30 days in situ using filter chambers allowing exposure to ambient groundwater, but not to ambient microorganisms. SMX-exposed UAC bacterial communities displayed the greatest mortality and impairment (viable stain assays), the greatest change in sensitivity to SMX (dose–response assays), and the greatest change in community composition (Terminal Restriction Fragment Length Polymorphism; T-RFLP). The sul1 gene, encoding resistance to SMX at clinically relevant levels, and an element of Class I integrons, was not detected in any community. Changes in microbial community structure and SMX resistance over a short experimental period in previously nonexposed, slow-growing aquifer communities suggest concentrations of antibiotics 2–3 orders of magnitude less than those used in clinical applications may influence ecological function through changes in community composition, and could promote antibiotic resistance through selection of naturally resistant bacteria.