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Microbial electrosynthesis (MES) is a biocathode-driven process, producing high-value chemicals from CO 2 . Here, an in situ self-assembled graphene oxide (rGO)/biofilm was constructed, in MES, for high efficient acetate production. GO has been successfully reduced by electroautotrophic bacteria for the first time. An increase, of 1.5 times, in the volumetric acetate production rate, was obtained by self-assembling rGO/biofilm, as compared to the control group. In MES with rGO/biofilm, a volumetric acetate production rate of 0.17 g l −1 d −1 has been achieved, 77% of the electrons consumed, were recovered and the final acetate concentration reached 7.1 g l −1 , within 40 days. A three-dimensional rGO/biofilm was constructed enabling highly efficient electron transfer rates within biofilms, and between biofilm and electrode, demonstrating that the development of 3D electroactive biofilms, with higher extracellular electron transfer rates, is an effective approach to improving MES efficiency. [ABSTRACT FROM AUTHOR]