Biofilm formation is inevitable in a bioelectrochemical system in which microorganisms act as a sole biocatalyst. Cathodic biofilm (CBF) works as a double-edged sword in the performance of the air-cathode microbial fuel cells (MFCs). Proton and oxygen crossover through the CBF are limited by the robust structure of extracellular polymeric substances, composition of available constituents and environmental condition from which the biofilm is formed. The MFC performance in terms of power, current and coulombic efficiency is influenced by the nature and origin of CBF. Development of CBF from different ecological environment while keeping the same anode inoculums, contributes additional charge transfer resistance to the total internal resistance, with increase in coulombic efficiency at the expense of power reduction. This study demonstrates that MFC operation conditions need to be optimized on the choice of initial inoculum medium that leads to the biofilm formation on the air cathode.