A novel mixotrophic biofilm reactor (MBR) was operated to simultaneously remove total organic carbon, nitrate, and Mn(II). Effects of some key factors on mixotrophic denitrification were investigated in lab-scale bioreactors. Optimized conditions obtained from the MBR were used to validate the experiments, proving their feasibility. High-throughput sequencing was employed to analyze the microbial communities of the MBR. The community diversity in the MBR was markedly influenced by the ratio of Cinorganic: Corganic. The species were increased with lower Cinorganic: Corganic concentration. Pseudomonas was detected as the dominant species in the MBR, indicating that Pseudomonas sp. SZF15 and Pseudomonas sp. H-117 play a primary role in the process of nitrate removal.