The development of microbial fuel cells based on electro-catalytic processes is among the novel topics, which are recently emerging in the sustainable development of energetic systems. Microbial fuel cells have emerged as unique biocatalytic systems, which transform the chemical energy accumulated in renewable organic fuels and at the same time reduce pollution from hazardous organic compounds. However, not all microorganisms involved in metabolic/catalytic processes generate sufficient redox potential. In this research, we have assessed the applicability of the microorganism Rhizobium anhuiense as a catalyst suitable for the design of microbial fuel cells. To improve the charge transfer, several redox mediators were tested, namely menadione, riboflavin, and 9,10-phenanthrenequinone (PQ). The best performance was determined for a Rhizobium anhuiense-based bio-anode mediated by menadione with a 0.385 mV open circuit potential and 5.5 μW/cm² maximal power density at 0.35 mV, which generated 50 μA/cm² anode current at the same potential.