Abstract: The construction of the Three Gorges Dam has significantly altered water and sediment fluxes in the Yangtze River, yet its impact on microbially mediated multifunctionality (carbon, nitrogen, and phosphorus cycling) in sediments remains unclear. This study employed 16S amplicon sequencing, co-occurrence network analysis, and structural equation modeling to investigate the effects of the dam on bacterial community composition, diversity, and multifunctionality in upstream and downstream sediments. Results revealed that upstream sediments were dominated by Proteobacteria (~25.5%), while downstream sediments were dominated by Desulfobacterota (~24.8%). The dam significantly reduced bacterial α-diversity in downstream sediments, which was closely correlated with reductions in moisture (~35.4%), cation exchange capacity (~49.6%), and dissolved organic carbon (~49.9%). Structural equation modeling indicated that reduced α-diversity directly impaired ecological functions and indirectly decreased multifunctionality by weakening bacterial network interactions. This study highlights the adverse ecological impacts of the Three Gorges Dam on downstream sediment functions and recommends long-term monitoring of dissolved organic carbon and cation exchange capacity, alongside optimized reservoir management, to mitigate biodiversity loss.