Correlations among biofilm activity, chemistry and power production of membraneless, single chamber microbial fuel cells (MFC) were established using four microelectrodes. Each different (pH, redox, conductivity, S-2) microelectrode was assembled, calibrated and located close to the cathode. Power productivity of five MFCs was explained in terms of response of the microelectrodes. pH variation demonstrated that a proton gradient establishes within the cathodic biofilm, increasing acidity near the electrode. Conductivity increases inside the biofilm, proving low diffusion and increased ion concentration. Redox profiles provide a significant improvement to the understanding of the biochemical equilibria inside and outside the biofilm. Sulphide variations emphasize the role of the sulphur cycle in the MFC development. Diffusion hindrance seems the key-factor for the development of a biofilm and the establishment of a natural separation of the cell in cathodic and anodic compartments. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.