A key criterion in microbial fuel cell (MFC) design is that the bio-electrochemical reaction between bacteria and the bulk solution should occur evenly on the electrode surface in order to improve electricity generation. However, experimental optimization of MFC design over a wide range of conditions is limited. Computational fluid dynamics (CFD) technology makes it possible to evaluate physicochemical phenomena such as fluid flows, mass transfer and chemical reaction, which can assist in system optimization. Twelve MFCs (M1–M12) with different internal structures were subjected to CFD analysis. The dead (DS) and working spaces (WS) of the anode compartment were calculated. The flow patterns of the anodic fluid varied according to the internal structures. The WS where the bio-electrochemical reaction can actually occur varied over the range of 0.14–0.57 m2. Based on the above results, the power densities were estimated under the assumption that a monolayer biofilm was formed on the electrode. M11, with 18 rectangular-type internal structures, showed the largest WS of 0.57 m2 and a theoretical maximum power density of 0.54 W/m2. Although the optimization of the MFC configuration with only CFD analysis remains limited, the present study results are expected to provide fundamental data for MFC optimization.