Exogenous electron acceptors, such as nitrate, hold great potential for the bioremediation of wastewater contaminated with bromophenols (BPs). However, research into the mechanisms underlying BPs biodegradation remains in its early stages, particularly regarding the molecular structure and bioremediation performance. This study provides a comprehensive analysis of the mechanisms involved in BPs within a nitrate-reducing system, focusing on the molecular structure of BPs. Therefore, three up-flow bioreactors were operated for 187 days, achieving removal efficiencies of 100 %, 90.4 ± 0.6 % and 50.2 ± 2.8 % for ortho-bromophenol (2-BP), para-bromophenol (4-BP) and meta-bromophenol (3-BP), respectively. Hydrolytic dehalogenase (LinB) was found to play a critical role in BP metabolism. Molecular docking and density functional theory calculations revealed that the geometric structure and electronic effects of the Br-substituent significantly influenced LinB activity and BP reactivity, thereby affecting removal efficiencies. Notably, 2-BP, with a shorter orientation distance, was more readily catalyzed by LinB, as evidenced by metagenomic analyses showing significant increases in the abundance of N-transforming and BP-degrading genes. Furthermore, 2-BP and 4-BP stimulated more robust microbial responses, including dehalogenation (Thauera), denitrification (Delftia), and electron transport (Xanthomonadales). These results provide valuable insights into the environmental fate of BPs at the molecular level and how the Br-substituent influences microbial metabolism.