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In this study, the nitrogen-fixing, Gram-negative soil bacteria <i>Rhizobium anhuiense</i> was successfully utilized as the main biocatalyst in a bacteria-based microbial fuel cell (MFC) device. This research investigates the double-chambered, H-type <i>R. anhuiense</i>-based MFC that was operated in modified Norris medium (pH = 7) under ambient conditions using potassium ferricyanide as an electron acceptor in the cathodic compartment. The designed MFC exhibited an open-circuit voltage (OCV) of 635 mV and a power output of 1.07 mW m⁻² with its maximum power registered at 245 mV. These values were further enhanced by re-feeding the anode bath with 25 mM glucose, which has been utilized herein as the main carbon source. This substrate addition led to better performance of the constructed MFC with a power output of 2.59 mW m⁻² estimated at an operating voltage of 281 mV. The <i>R. anhuiense</i>-based MFC was further developed by improving the charge transfer through the bacterial cell membrane by applying 2-methyl-1,4-naphthoquinone (menadione, MD) as a soluble redox mediator. The MD-mediated MFC device showed better performance, resulting in a slightly higher OCV value of 683 mV and an almost five-fold increase in power density to 4.93 mW cm⁻². The influence of different concentrations of MD on the viability of <i>R. anhuiense</i> bacteria was investigated by estimating the optical density at 600 nm (OD₆₀₀) and comparing the obtained results with the control aliquot. The results show that lower concentrations of MD, ranging from 1 to 10 μM, can be successfully used in an anode compartment in which <i>R. anhuiense</i> bacteria cells remain viable and act as a main biocatalyst for MFC applications.