Electrochemical impedance spectroscopy, cyclic voltammetry, and polarization tests were used to monitor the progress of the anode colonization by electrode-reducing microorganisms in a single-chamber membraneless microbial fuel cell seeded with anaerobic sludge. The electrochemical methods showed that an increase in microbial fuel cell power output coincided with a progressive decrease of the anode internal resistance and a more negative open circuit potential. Two redox systems were observed in cyclic voltammograms shortly after microbial fuel cell startup, while a redox system with a peak around -330 mV (vs. Ag/AgCl) was predominant in the mature biofilm. The redox systems were also dependent on the external resistance chosen for microbial fuel cell operation. This suggests that within the diverse microbial populations several species are capable of electron transfer to the anode, and that the microorganisms with the highest electron transfer rate become predominant. Furthermore, the growth of these electrode-reducing microorganisms can be accelerated by optimizing the microbial fuel cell electrical load.