Microbial fuel cells (MFCs) hold great promise as sustainable bioenergy sources, with their performance intricately linked to the formation and characteristics of biofilms. This study delves into the bio-electrochemical perspective of biofilms in MFCs, aiming to elucidate their pivotal role in MFC functionality. The investigation focused on a yeast-based MFC operated through 48 h per cycle, with cycle 5 marking the maturation stage of biofilm formation. During this phase, voltage stability was observed, with a stationary phase voltage of 38.9±2.6 mV. Notably, cycle 5 exhibited a significant boost in power density, reaching 8.82 mW m-2, accompanied by the lowest internal resistance of 100 Ω. Furthermore, the electron transfer rate constant from cycle 5 is 1.14±0.02 s-1, 57 times higher than the initial, underscoring biofilm's catalytic potential. Additionally, cyclic voltammetry unveiled non-linear relationships between redox reaction peak current and scan rate, with a consistent DEp of ~219 mV at 100 mV s-1. Importantly, elemental analysis disclosed incorporating diverse elements (Na, Al, Si, P, S, Cl, K, Ca, Cr, and Fe) into the carbon felt, signifying their association with biofilm development. These findings offer critical insights into optimizing MFC performance through biofilm modulation, advancing sustainable bioenergy technologies.