The production of bioelectricity has become an urgent need in the present global context due to its renewable characteristics, sustainability, and potential to alleviate the energy crisis while positively contributing to environmental conservation. The Microbial Fuel Cell (MFC) technology is recognized as a promising method for the production of sustainable electricity and aiding in the environmental restoration. Ongoing research aims to enhance MFC efficiency by improving electrode materials and optimizing microbial interactions. In this context, microbes-mediated enzymatic degradation and bioconversion of organic-rich wastewater are important factors in carrying out the proper functioning of MFC and controlling environmental pollution as well. Exploring the mechanistic role of source microbiota in bioelectricity production from wastewater is particularly interesting, as these systems involve complex biochemical processes and microbial interactions within microbial electrochemical systems (MES). During the breakdown of organic pollutants through microbial interactions, chemical energy is converted into electrical energy via various electrochemical reactions. Therefore, an in-depth understanding of microbial populations and the dynamics of their metabolic reactions could be a central focus for optimizing the bioelectricity production from organic pollutant-rich wastewater. In this book chapter, we thoroughly discuss the nature and mechanistic role of source microbiota related to bioelectricity generation from organic compound-rich wastewater. The chapter will cover the intricate interplay of microbial enzymatic reactions and major metabolic pathways, highlighting the core processes of MFC. Understanding these processes at the molecular level is crucial for advancing the development and optimization of efficient bioenergy production.