Microbial fuel cells (MFCs) have garnered significant interest from researchers as a self-sustaining and environmentally friendly system for the simultaneous treatment of urban and industrial wastewater and production of bioelectricity. The type and material of the electrode are critical factors that can influence the efficiency and energy production of this treatment process, with porosity, surface area, conductivity, and stability being key considerations. In this study, graphite plates and carbon felt were modified through the electrodeposition of nickel followed by the formation of a biofilm, resulting in conductive bio-anode thin film electrodes with enhanced power generation capacity. The structural and morphological properties of the electrode surfaces were characterized using X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), elemental mapping, and field-emission scanning electron microscopy (FE-SEM) techniques. The results confirmed that nickel was successfully doped and that sufficient amounts of microorganisms were attached to the nickel-deposited electrodes, leading to improved electron transfer and increased power generation. To validate this claim, various parameters such as maximum voltage, current density, and power generation were investigated using a dual-chamber MFC equipped with a Nafion 117 membrane and bio-nickel-doped carbon felt (bio-Ni@CF) and bio-nickel-doped graphite plate (bio-Ni@GP) electrodes under constant temperature conditions. The polarization curve obtained during four loading stages using different anode electrodes indicated that the maximum voltage achieved was 468.0 mV using the bio-Ni@CF electrode, representing an increase of 35.0%, 18.37%, and 9.82% compared to the bare GP, bare CF, and bio-Ni@GP electrodes, respectively. The highest power density and current density achieved using the bio-Ni@CF electrode were 130.72 mW/m2 and 760.0 mA/m2, respectively. Furthermore, the modified electrodes demonstrated appropriate stability and resistance during successful runs. These results suggest that nickel-doped carbon-based electrodes can serve as suitable and stable supported catalysts and conductors for improving efficiency and increasing power generation in MFCs.