The main limiting factor in Microbial Fuel Cell (MFC) power output is the cathode, because of the high overpotential for oxygen reduction. Oxygen reducing biocathodes can decrease this overpotential by the use of microorganisms as a catalyst. In this study, we investigated the factors limiting biocathode performance. Three biocathodes were started up at different cathode potentials, and their performance and catalytic behavior was tested by means of polarization curves and cyclic voltammetry. The biocathodes controlled at +0.05 V and +0.15 V vs Ag/AgCl produced current almost immediately after inoculation, while the biocathode controlled at +0.25 V vs Ag/AgCl produced no current until day 15. The biocathode controlled at +0.15 V vs Ag/AgCl reached the highest current density of 313 mA/m2. Cyclic voltammetry showed clear catalysis for all three biocathodes. The biocathodes were limited by both mass transfer of oxygen and by charge transfer. Mass transfer calculations show that the transfer of oxygen poses a serious limitation for the use of dissolved oxygen as an electron acceptor in MFCs.