Simultaneous decolorization and mineralization of high concentrations of methyl orange (MO) by mixed microbial culture via denitrification pathway was studied in a 1.5 L capacity anoxic up-flow packed bed reactor. Feasibility of dye degradation at varying concentrations of MO, sucrose, and NO3−-N were studied with varied hydraulic retention time (HRT). The mixed microbial culture procured from a sequencing batch reactor treating domestic sewage exhibited a high affinity for decolorization/mineralization of MO up to 500 mg/L. Optimum removal of 97 % of color, 88 % of TN, 86 % of COD, and 83 % of TOC was observed at HRT of 12 h. Decolorization followed a first-order kinetics with rate constant of 0.1/h. The presence of sucrose and NO3−-N at optimal concentrations significantly enhanced MO decolorization/mineralization. The optimal COD/NO3−-N ratio for effective simultaneous decolorization and mineralization of 500 mg/L MO at 12 h HRT was 48. UV–vis absorption spectra of effluent and TOC data indicate effective decolorization and mineralization of dye, respectively. Hence the developed process shows the potential of simultaneous removal of color, COD/TOC, and TN effectively in a single reactor. However, further research is required to explore the application of this process in treating real textile dye effluent having mixed dyes. © 2020 Elsevier Ltd