No studies have reported the operation optimization of constructed wetland-microbial fuel cell (CW-MFC) systems in terms of pollutant removal under the influence of multiple factors. Multifactor orthogonal experiment (L25(55)) was designed in this study to investigate the influence of multiple factors on the CW-MFC performance and determine the optimal operating conditions for the organics and nutrient removal. The tested factors include volume ratio of granular graphite in the substrates (A), dissolved oxygen (DO) concentration in the cathode zone (B), hydraulic retention time (HRT) (C), effluent reflux ratio (D), and external resistance (E). The results showed that the sequence and degree of the influence of the tested factors were C** > B** > E** > D* > A for chemical oxygen demand (CODCr) removal, C** > B** > D* > E > A for ammonia nitrogen (NH3-N) removal, C** > D** > B** > E* > A* for total nitrogen (TN) removal, and C** > D* > B > A > E for total phosphorus (TP) removal (* denotes significant influence (0.01 < p < 0.05) and ** denotes extremely significant influence (p ≤ 0.01)). HRT was found to be the most influential factor for pollutant removal in CW-MFCs with a contribution of over 50% for CODCr, NH3-N and TP removal, and over 45% for TN removal. The optimal operating conditions for CODCr, NH3-N, TN and TP removal in CW-MFCs were quite different from each other. Comprehensively considering the treatment efficiency of pollutant, treatment capacity of wastewater, and energy consumption from artificial aeration, the selected comprehensive optimal operating conditions for CW-MFCs were A = 20%, B = 1.5 mg/L, C = 1.5 days, D = 50%, and E ≤ 250 Ω. Moreover, incorporating the MFC significantly enhanced the organics and nitrogen removal in CWs by 8.72-11.04% CODCr and 9.78-12.04% TN.