Abstract: Existing fluoride removal methods still suffer from limitations in cost, stability, and adaptability to complex water systems, making the development of closed-loop mechanisms with low energy consumption a critical challenge. A microbial electrolysis cell (MEC) coupled with an anaerobic biofilm reactor was constructed to achieve directional enrichment, transformation, and fixation of fluoride in mine water through synergistic effects of applied potential and biofilm, while systematically analyzing microbial community characteristics and functional genes during defluoridation. Results showed that fluoride concentration exhibited rapid initial reduction followed by gradual stabilization, with solid-phase composition shifting from carbonates/sulfides to fluoride-dominated phases (enhanced NaF signals), and overall weakening of liquid-phase peaks related to C—F bonds, hydroxyl groups, aliphatic C—H, and oxygen-containing functional groups. In liquid organic matter, multiple fluorine-containing compounds (C1—C30) were detected by MEC and biofilm reactors in positive/negative ion modes, indicating continuous organic consumption and transformation. Microbial communities were dominated by Pseudomonadota at the phylum level, with Sphingomonas, Methyloversatilis, and Aquabacterium as predominant genera. Metagenomic analysis revealed that the dominant metagenome-assembled genomes (MAGs) commonly harbor three major functional gene clusters involved in organic pollutant degradation metabolism (dhlA and fadA), electron transfer (cob/cbi and nuo), and mineralization and fluoride tolerance (phoABRX, pstABCS and crcB), collectively supporting a sequential pathway from organic activation to defluorination and ultimately to inorganic fixation.Electrobiological coupling-driven mechanisms for the directional enrichment, cleavage, and mineralization-immobilization of fluoride, providing a basis for developing low-consumption, stable-output synergistic defluoridation strategies in complex mine water systems.