The mixotrophic denitrification desulfurization process was adopted in an upflow attached-growth bioreactor for wastewater treatment. The microorganisms in the system were acclimated to remove sulfide, nitrate, nitrite, and organics simultaneously while the contaminants were finally converted to element sulfur, nitrogen gas, and carbon dioxide. The 16S rDNA clone library technique was utilized to investigate the characteristics of the microbial community within this mixotrophic environment. The results showed that Azoarcus was the main sulfur-based denitrification desulfurization bacteria and had a relative abundance of 8.0% in this system. It used sulfide as electron donors, with both nitrate and nitrite as eletron acceptors. Thauera, Vulcanibacillus, and Paracoccus were the main heterotrophic denitrification bacteria and had the relative abundances of 29.5%, 9.82%, and 1.78%, respectively. They utilized arganics as electron donors, with nitrate or nitrite as electron acceptors. The high removal efficiencies of contaminants were attributed to the interaction of these two main species.