In order to investigate the impact of sulfadiazine (SDZ) on the functionality and structure of electroactive biofilms, a combination of electrochemical performance monitoring with microbial community structure analysis was used to elucidate its mechanisms in microbial fuel cells (MFCs) under different SDZ concentrations (0, 0.5, and 10 mg/L). The results show that 0.5 mg/L SDZ significantly enhance the current output and electrochemical activity of MFCs by facilitating extracellular electron transfer. In contrast, 10 mg/L SDZ markedly inhibits MFC performance and exerts toxic effects on electroactive microbial communities. SDZ exposure induces significant shifts in the dominant microbial populations and electron transfer pathways within the biofilm. It is concluded that sulfadiazine exerts a concentration-dependent influence on MFC performance, whereby appropriate levels may optimize electrochemical behavior, while high concentrations can threaten biofilm stability and ecological function.