A nickel (Ni) nanoparticles- (NPs) dispersed web of carbon micro-nanofibers (ACFs/CNFs) was fabricated as the electrode of a microbial fuel cell (MFC) for bio-energy production using Escherichia coli as a microbial catalyst. The multiscale web of ACFs/CNFs was prepared using chemical vapor deposition, with the Ni NPs as the catalyst and benzene as the carbon source to grow the CNFs on the ACF substrate. The Ni NPs attached at the tips of graphitic CNFs facilitated the transfer of E. coli-released electrons to the anode of MFCs. Linear sweep voltammetry was performed to determine power density and open circuit potential (OCP) of the prepared MFCs. The power density and OCP were experimentally measured as 710 ± 5 mV and 1145 ± 20 mW/m2, respectively, which were approximately 9 times greater than those of the ACF substrate-based MFCs. The transition metal-CNFs-based electrodes prepared in this study may be a potentially alternative to the expensive noble metals-based electrodes presently used in MFCs.