The tobacco production process generates a substantial amount of wastewater characterized by high organics and low biodegradability, which poses a significant risk of severe environmental pollution. In order to explore a clean and low-cost technology for tobacco wastewater treatment, this study constructed two-chamber MFCs and investigated the performance of tobacco wastewater treatment and electricity generation capacity at room temperature. The incorporation of carbon sources (e.g., glucose, acetate, propionate, and butyrate) in wastewater could enhance the removal of COD, total nitrogen and ammonia nitrogen in wastewater. After three cycles, the maximum COD removal rate reached 75.97 +/- 1.49%, while the maximum total nitrogen removal and ammonia nitrogen removal rates were 46.95 +/- 1.77% and 48.31 +/- 1.16%, respectively. Meanwhile, the maximum voltage output of 0.67 V was observed, and the maximum power density was 717.04 mW/m(2.) The microbial community analysis revealed that Trichococcus and Acinetobacter were present in high abundance in MFCs, which may play a significant role in electricity generation and wastewater treatment. These results demonstrate that MFC is applicable for tobacco wastewater treatment, providing both theoretical foundation and technical references for the large-scale practical application of MFC technology in tobacco wastewater treatment.