In this study, the effect of utilization of a nanostructured nickel based material as a negative electrode on the performance of microfluidic microbial fuel cell (MFC) with Escherichia coli as biocatalyst has been investigated. Designing the microfluidic MFC with nickel nanostructure resulted in a higher volumetric power density of 343 W m−3 compared to the previously published results. The assessment of effective parameters on the electrochemical performance of cell was investigated. The investigation of the hydraulic diameter impact on the power generation proves that reducing the microchannel hydraulic diameter from 1000 to 350 μm minimized the internal mass-transfer resistance, and consequently 32% increase in the maximum surface power density is observed. Replacing the nickel foil with nickel nanostructures resulted in an additional improvement of 67% in the maximum surface power density. The maximum volumetric power density of 343 W m−3 has been obtained for the fabricated MFC with 350 μm hydraulic diameter associating nanostructured morphology. To assess the capability of the microfluidic MFC as a power source of the energy storage devices, the obtained results show that the microfluidic MFC with nickel nanostructure is able to provide about 76% of the total power needed for manganese rechargeable lithium battery.