Sulfamethoxazole (SMX) is an extensively consumed sulfonamide antimicrobial agent and is frequently detected in surface water. This work studied the degradation process of SMX in anodic chamber of microbial fuel cell (MFC) reactors. We found that the biodegradation of SMX could be achieved after acclimation and even high concentrations of SMX (e.g. 200 ppm) could be rapidly degraded. Excitation and emission matrix fluorescence spectroscopy analysis revealed that the chemical structure of SMX was altered during the process. Q-Exactive hybrid quadrupole-Orbitrap mass spectrometry was used to identify the degradation byproducts of SMX. The activity of electrode biofilm was examined afterwards and it was found that the microbe was in an active state. High-throughput sequencing analysis suggested that the microbial community structure was greatly changed during the process; some reported SMX scavengers, such as Achromobacter and Pseudomonas, were abundant in the reactors. Some metazoans were also recognized in the biofilm samples, which indicates that the operation of the MFC reactors was in a steady state. This study discusses the degradation mechanism of SMX and explores the microbial community response during the process, which provides useful information for the application of MFC in antibiotic elimination.