The development and the following inactivation of a carbon-based biocathode in single chamber and membraneless MFCs was investigated in this work. The electrochemical behavior of the biocathode has been analyzed over time during the MFC life. X-ay Micro-Computed Tomographies (microCTs) have been carried out at progressive stages, documenting the building over time of a layer of scale deposition becoming thicker and thicker up to the cathode inactivation. The technique provides cross-sectional (tomographic) grayscale images and 3D reconstruction of volumes. Lighter color indicates lower X-ray attenuation (i.e., lower atomic density) thus allowing distinguishing biofilm from inorganic fouling on the basis of the average atomic number Z of each voxel (3D pixel). MicroCT was combined with Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Spectroscopy (EDX) in order to qualitatively recognize chemical species in each different layer of the cathode's section. Results correlated the presence of biofilm and calcium carbonate deposits, prevalently in the inner part of the cathode, with the produced electric current over time. A specific microCT-related software quantified the time-dependent carbonate scale deposition, identifying a correlation between the decreasing performances of the device and the increasing quantity of scale deposition that penetrates the cathode cross section in time. (C) 2017 Elsevier B.V. All rights reserved.