A novel and integrated biofilm process – the sulfate reduction (SR) and mixotrophic (MR) sulfide-utilization denitrification process (SMSD) – was recently proposed for sulfate treatment and sulfur recovery. The process consisted of two bioreactors: a 5.1 L anaerobic upflow reactor for SR, and a 3.5 L anaerobic upflow reactor for MR desulfurization–denitrification. The experiment was conducted for 370 days to evaluate the performance of SMSD at various sulfate concentrations and hydraulic retention times. The process successfully achieved sulfate, organics and nitrogen compound removal efficiencies of 94.1, 97.7 and 99.1%, respectively. Sulfate was predominantly converted to element sulfur, while nitrate and nitrite were finally converted to nitrogen gas. In SR, with the help of high pH and sponge cubes with various bacteria, 97.5% of sulfide conversion efficiency and 540 mgS/L of sulfide were obtained. In MR, sulfide was removed up to 100% and was partially oxidized to sulfur. The extent of heterotrophic denitrification, which ranged from 35.8 to 59.8%, depended on the categories of electron acceptors.