We used a hydrogen (H2)-based biofilm to treat a groundwater contaminated with perchlorate (ClO4−) at ∼10 mg/L, an unusually high concentration. To enhance ClO4− removal, we either increased the H2 pressure or decreased the electron-acceptor surface loading. The ClO4− removal increased from 94% to 98% when the H2 pressure was increased from 1.3 to 1.7 atm when the total acceptor surface loading was 0.49 g H2/m2 day. We then decreased the acceptor surface loading stepwise from 0.49 to 0.07 g H2/m2 day, and the ClO4− removal improved to 99.6%, giving an effluent ClO4− concentration of 41 µg/L. However, the tradeoff was that sulfate (SO42−) reduction occurred, reaching 85% conversion at the lowest acceptor surface loading (0.07 g H2/m2 day). In two steady states with the highest ClO4− reduction, we assayed for the presence of perchlorate-reducing bacteria (PRB), denitrifying bacteria (DB), and sulfate-reducing bacteria (SRB) by quantitative polymerase chain reaction (qPCR) targeting characteristic reductases. The qPCR results documented competition between PRB and SRB for space within the biofilm. A simple model analysis for a steady-state biofilm suggests that competition from SRB pushed the PRB to locations having a higher detachment rate, which prevented them from driving the ClO4− concentration below 41 µg/L. Biotechnol. Bioeng. 2013;110: 3139–3147. © 2013 Wiley Periodicals, Inc.