With much of the MFC research around the world being focused on the engineering of the reactors, understanding the fundamental relationship between the microbiology and electrochemistry is critical to the advancement of this technology. Biofilms of variable thickness were grown separately in different microbial fuel cell reactors which were subjected to extensive electrochemical and microbiological tests. The correlation between microbial biofilm properties and the electrochemical performance was investigated in detail. It was found that the cell count, biofilm density and the type of communities in the anode biofilm had a direct influence on the power output of microbial fuel cells. Also the power density was directly dependent on the biofilm growth and increased significantly during the initial biofilm growth period. A variation in the biofilm morphology and composition was observed during the biofilm growth period which may have a serious impact on the performance of the microbial fuel cells.