Microbial fuel cells (MFCs) are bio-electrochemical transducers that produce electrical energy by the decomposition of organic matter with the aid of microorganisms. Through the transfer of electrons, energy can be delivered to an electrode surface, and understanding the electron transport mechanisms has become very crucial. Although significant research has been carried out in this field, relatively few reports have been based on numerical simulations. Therefore, this study was initiated to design a computational model through numerical simulation and apply it to the betterment of MFCs. Three important biochemical mechanisms of MFCs such as direct electron transport, transport through electron shuttles, and transport through nanowires were considered for simulation. The results showed that the function of the thickness of the active biofilm (jmax) was obtained at a substrate concentration of 1.1 M with a current of 0.16 mA. The direct electron transport mechanism was reported to produce the maximum current density of 15.14 mA/m2. The direct transport also used a higher concentration of substrate to generate power than the nanowire transport and electron shuttle processes. These findings provide useful information on the enhancement of the performance of MFCs and especially on the application of numerical simulations for their scale-up process.