This study aims to investigate the effect of the graphite felt (GF) substrate surface treatment, the GF active surface area, and the anode chamber depth on the performance of the passive air breathing flat-plate microbial fuel cell (FPMFC) configuration. Three passive air breathing FPMFCs (depth of anode chamber: 2mm, 4mm, and 8mm) were developed and operated using 1, 2, and 3 packed layers of three-dimensional (3D) graphite felt anodes, respectively, with similar cross sectional (geometric) surface area as the cathode and the membrane. The surface of the GF substrate was treated by soaking in a hot solution of nitric acid prior to inoculation. The 2mm FPMFC generated a peak power density superior to that previously reported for the same configuration with no GF treatment. The peak power density in the 8mm and 4mm FPMFCs with 3 and 2 layers of GF increased by 118 % and 48 %, respectively, compared to the 2mm FPMFC with 1 layer of GF. By using only 1 layer of GF, the peak power density showed no significant variation with the electrode spacing.