ABSTRACTThe aim of this study was to characterize the pollutant removal efficiency and the microbial communities that arose in a newly designed waterfall biofilm reactor (WFBR) at different chemical oxygen demand/total nitrogen (COD/TN) ratios. The reactor was operated continuously for 28 days at different COD/TN ratios, and its efficiency was evaluated. Results showed that as the thickness of the biofilm increased, the structure of the biofilm encouraged anaerobic-aerobic, anoxic-anaerobic, and fully anaerobic conditions in one reactor. The COD/TN ratios used had a significant effect on the removal of COD and nitrogen components. At a COD/TN ratio of 14, the ammonium nitrogen removal efficiency reached its highest value (99%), but the COD removal efficiency remained at approximately 90%. High-throughput sequencing revealed that the highest community diversity and richness were seen at a COD/TN ratio of 18, and the major phyla were Proteobacteria (average abundance of 47%), Actinobacteria (24%), and Bacteroidetes (13%). As the COD/TN ratios increased from 7 to 18, the abundance of Proteobacteria gradually increased from 25% to 68%. These results could provide important guidance for the design of new wastewater treatment systems and also enrich our theoretical understanding of microbial ecology.