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The structure and activity of electrochemically active biofilms (EABs) are usually investigated on flat electrodes. However, real world applications such as wastewater treatment and bioelectrosynthesis require tridimensional electrodes to increase surface area and facilitate EAB attachment. The structure and activity of thick EABs grown on high surface area electrodes are difficult to characterize with electrochemical and microscopy methods. Here, the authors adopt a stacked electrode configuration to simulate the high surface and the tridimensional structure of an electrode for large-scale EAB applications. Each layer of the stacked electrode is independently characterized using confocal laser scanning microscopy (CLSM) and digital image processing. Shewanella oneidensis MR-1 biofilm on stacked carbon veil electrodes is grown under constant oxidative potentials (0, þ200, and þ400mV versus Ag/AgCl) until a stable current output is obtained. The textural, aerial, and volumetric parameters extracted from CLSM images allow track- ing of the evolution of morphological properties within the stacked electrodes. The electrode layers facing the bulk liquid show higher biovolumes compared with the inner layer of the stack. The elec- trochemical performance of S. oneidensis MR-1 is directly linked to the overall biofilm volume as well as connectivity between cell clusters.