Air cathode microbial fuel cells (MFCs) were used in a cascade-system, to treat neat human urine as the fuel. Their long-term operation caused biodeterioration and biofouling of the cathodes. The cathodes were made from two graphite-painted layers, separated by a current collector. The initial performance of the MFCs was reaching average values of 105.5 ± 32.2 μW and current of 1164.5 ± 120.2 μA. After 3 months of operation the power performance decreased to 9.8 ± 3.5 μW, whilst current decreased to 461.2 ± 137.5 μA. Polarisation studies revealed significant transport losses accompanied by a biofilm formation on the cathodes. The alkaline lysis procedure was established to remove the biomass and chemical compounds adsorbed on the cathode's surface. As a result, the current increased from 378.6 ± 108.3 μA to 503.8 ± 95.6 μA. The additional step of replacing the outer layer of the cathode resulted in a further increase of current to 698.1 ± 130 μA. Similarly, the power performance of the MFCs was recovered to the original level reaching 105.3 ± 16.3 μW, which corresponds to 100% recovery. Monitoring bacterial cell number on the cathode's surface showed that biofilm formed during operation was successfully removed and composed mainly of dead bacterial cells after treatment. To the best of the authors' knowledge, this is the first time that the performance of deteriorating cathodes, has been successfully recovered for MFCs in-situ. Through this easy, fast and inexpensive procedure, designing multilayer cathodes may help enhance the range of operating conditions, if a biofilm forms on their surface.