This study aims at evaluating simultaneous chromate and nitrate reduction using sulfur-based mixotrophic denitrification process in a column reactor packed with elemental sulfur and activated carbon. The reactor was supplemented with methanol at C/N ratio of 133 or 2. Almost complete denitrification was achieved at influent NO3--N and Cr(VI) concentrations of 75 mg/L and 10 mg/L, respectively, and 3.7 h HRT. Maximum denitrification rate was 0.5 g NO3--N/(L.d) when the bioreactor was fed with 75 mg/L 150 mg/L methanol and 10 mg/L Cr(VI). The share of autotrophic denitrification was between 12% and 50% depending on HRT, C/N ratio and Cr(VI) concentration. Effluent total chromium was below 50 mu g/L provided that influent Cr(VI) concentration was equal or below 5 mg/L. DGGE results showed stable microbial community throughout the operation and the presence of sulfur oxidizing denitrifying bacteria (Thiobacillus denitrificans) and Cr(VI) reducing bacteria (Exiguobacterium spp.) in the column bed. (C) 2013 Elsevier B.V. All rights reserved.