The biological reduction of nitrate and perchlorate was comparatively evaluated in autotrophic and mixotrophic bioreactors using elemental sulfur and/or methanol as the energy source. The mixotrophic reactor was supplemented with methanol at CH3OH/NO3−-N ratio of 1 or 1.4. The mixotrophic reactor completely reduced perchlorate in the feed up to 1,000 μg l−1. The autotrophic reactor also showed high perchlorate reduction performance and decreased perchlorate from 1,000 μg l−1 to around 33 μg l−1. Complete reduction of 25 mg NO3−-N l−1 was achieved in both reactors, corresponding to a maximum nitrate reduction rate of 300 mg NO3−-N l−1d−1 and 400 mg NO3−-N l−1d−1 in the autotrophic and mixotrophic processes, respectively. Autotrophic denitrification caused an increase of effluent sulfate concentration, which may exceed the drinking water guideline value of 250 mg l−1. In the mixotrophic denitrification process, the effluent sulfate concentration was controlled by adjusting the C/N ratio in the influent. Mixotrophic denitrification was stimulated by 25 mg l−1 methanol addition and 53% of influent nitrate was reduced by the heterotrophic process, which decreased the effluent sulfate concentration to half of the autotrophic counterpart. Therefore, the mixotrophic process may be preferred over the autotrophic process when effluent sulfate concentration is of concern and a higher perchlorate reduction efficiency is desired.