We report an eco-friendly route for the reduction of graphene oxide (GO) by Azotobacter chroococcum at room temperature. Examinations using X-ray photoelectron spectroscopy, X-ray diffraction, and transmission electron microscopy indicate the formation of reduced graphene oxide (rGO) with a low degree of agglomeration. Quantitative analyses of electron diffraction data show that the produced rGO consists of single-layer sheets with a random stacking. It is suggested that the GO was reduced directly by the nitrogenase via sequential additions of electrons and protons followed by dehydration. This approach avoids the use of toxic chemical agents and also reduces the agglomeration of rGO. (C) 2017 Elsevier B.V. All rights reserved.