Harvesting electricity from the environment, organic wastes, or renewable biomass with microbial fuel cells (MFCs) is an appealing strategy, but the destructive sampling required to investigate the anode-associated biofilms has hampered research designed to better understand and optimize microbe–anode interactions. Therefore, a MFC that permits real-time imaging of the anode biofilm with confocal scanning laser microscopy was developed. In this new MFC Geobacter sulfurreducens, an organism closely related to those often found on MFC anodes and capable of high current densities, produced current comparable to that previously reported with other MFC designs. G. sulfurreducens engineered to produce the fluorescent protein mcherry to facilitate real-time imaging produced current comparable to wild-type cells. Introducing C-SNARF-4, a pH-sensitive fluoroprobe, into the anode chamber revealed strong pH gradients within the anode biofilms. The pH decreased with increased proximity to the anode surface and from the exterior to the interior of biofilm pillars. Near the anode surface pH levels were as low as 6.1 compared to ca. 7 in the external medium. Various controls demonstrated that the proton accumulation was associated with current production. Dropping the pH of culture medium from 7 to 6 severely limited the growth of G. sulfurreducens. These results demonstrate that it is feasible to non-destructively monitor the activity of anode biofilms in real time and suggest that the accumulation of protons that are released from organic matter oxidation within anode biofilms can limit current production.