In this work, four air-breathing microbial fuel cells (AB-MFC) were operated for 1 month in order to determine if the methodology of inoculation affects the steady-state performance of this type of MFCs. For this purpose, anaerobic and aerobic sludge were fed to two identical AB-MFCs without any external carbon source into a tight sealed environment during the first three days of start-up. For comparison purposes, other two AB-MFCs were operated mixing the initial sludge and an amount of sodium acetate as substrate. Results point out that the inoculation procedure does not affect the steady-state treatment capacity of the cells but it affects very seriously the production of electricity. Only the system fed with concentrated aerobic sludge was able to develop an efficient culture of bioelectrogenic microorganisms, while the other three systems failed in this purpose. This result was confirmed in a second independent series of experiments and by a last operation test, which consists of a re-inoculation of a low active MFC with microorganisms obtained from the best-performing cell in order to evaluate how much the MFC performance can be enhanced.