The production of low-pH effluent with sulfate and metals is one of the biggest environmental concerns in the mining industry. The biological process for sulfate reduction has the potential to become a low-cost solution that enables the recovery of interesting compounds. The present study analyzed such a process in a horizontal-flow anaerobic immobilized biomass (HAIB) reactor, employing ethanol as the carbon and energy source. Results showed that a maximal efficiency in the removal of sulfate and ethanol could only be obtained by reducing the applied sulfate load (225.1 ± 38 g m−3 d−1). This strategy led to over 75% of chemical oxygen demand (COD) and sulfate removal. Among the COD/SO42− studied ratios, 0.67 showed the most promising performance. The effluent's pH has naturally remained between 6.8 and 7.0 and the complete oxidation of the organic matter has been observed. Corrections of the influent pH or effluent recirculation did not show any significant effect on the COD and sulfate removal efficiency. Species closely related to strains of Clostridium sp. and species of Acidaminobacter hydrogenomorfans and Fusibacter paucivorans that can be related to the process of sulfate reduction were found in the HAIB reactors when the initial pH was 5 and the COD/SO42− ratio increased to 1.0.