The influence of Fe3O4 in proton conductivity, oxygen crossover, water absorption and performance of Sulfonated Poly Ether Ether Ketone (SPEEK) matrix was studied in single chamber microbial fuel cell at various weight percentages (2.5%, 5%, 7.5%, 10%) of SPEEK/Fe3O4. The fabricated composite membranes were characterized by FT-IR, SEM, and XRD which were also tested for their surface roughness, water absorption, proton conductivity and transportation of cations were also studied for its suitability in microbial fuel cell operation. Escherichia coil (E. coil), an electrogenic bacteria, was used as the biocatalyst in anodic chamber and platinized carbon (Pt/C) was used as the catalyst in the air cathode. The low oxygen crossover, selective transfer of protons and water mediated proton hopping mechanism in Fe3O4 composite membrane (7.5% Fe3O4) resulted in a maximum power density of 104 mW m(-2) which was higher than that of the power density of Nafion (R) 117 (47 mW m(-2)) under the same operating conditions. The results of the study suggested that SPEEK/Fe3O4 (7.5 wt%) composite membrane would be more commercially viable and efficient than the existing Nafion membranes for use in microbial fuel cells. (C) 2014 Elsevier B.V. All rights reserved.