This study focused on novel cathode structures to increase power generation and organic substrate removal in microbial fuel cells (MFCs). Three types of cathode structures, including two-layer (gas diffusion layer (GDL) and catalyst layer (CL)), three-layer (GDL, micro porous layer (MPL) and CL), and multi-layer (GDL, CL, carbon based layer (CBL) and hydrophobic layers) structures were examined and compared in single-chamber MFCs (SCMFCs). The results showed that the three-layer (3L) cathode structures had lower water loss than other cathodes and had a high power density (501 mW/m2). The MPL in the 3L cathode structure prevented biofilm penetration into the cathode structure, which facili- tated the oxygen reduction reaction (ORR) at the cathode. The SCMFCs with the 3L cathodes had a low ohmic resistance (Rohmic: 26e34 U) and a high cathode open circuit potential (OCP: 191 mV). The organic substrate removal efficiency (71e78%) in the SCMFCs with 3L cathodes was higher than the SCMFCs with two-layer and multi-layer cathodes (49e68%). This study demonstrated that inserting the MPL between CL and GDL substantially enhanced the overall electrical conduction, power generation and organic substrate removal in MFCs by reducing water loss and preventing biofilm infiltration into the cathode structure.