In a mediator-aided microbial fuel cell, the choice of a proper mediator is one of the most important factors for the development of a better fuel cell system as it transfers electrons from bacteria to the electrode. The electrochemical behaviors within the lipid layer of two representative mediators, thionin and safranine O both of which exhibit reversible electron transfer reactions, were compared with the fuel cell efficiency. Thionin was found to be much more effective than safranine O though it has lower negative formal potential. Cyclic voltammetric and fluorescence spectroscopic analyses indicated that both mediators easily penetrated the lipid layer to pick up the electrons produced inside bacteria. While thionin could pass through the lipid layer, the gradual accumulation of safranine O was observed within the layer. This restricted dynamic behavior of safranine O led to the poor fuel cell operation despite its good negative formal potential.