Trichloroethene (TCE) is a commonly used chlorinated compound that has been proven to be harmful to humans and the environment. TCE dechlorination under anaerobic conditions is an applicable and cost-effective treatment method to cleanup TCE-polluted groundwater. Supplement of organic substrates could enhance the anaerobic production of hydrogen, which could improve the activity of dechlorinating bacteria [Dehalococcoides (DHC)]. However, the produced hydrogen would be consumed by methanogens and result in decreased DHC growth efficiency and TCE dechlorinating rate. In this microcosm study, different methanogen inhibitors [2-bromoethanesulfonate (BES), 2-chloroethanesulfonate (CES), molybdate (Mo), and Mo plus BES] were applied for methanogens growth inhibition. The Clostridium butyricum (hydrogen-producing bacterium) was supplied in the microcosm system to improve the hydrogen production efficiency. Enhanced TCE removal (up to 92% of TCE dechlorination) and DHC growth (increased from 5.7 × 103 to 1.1 × 105 gene copies/mL) efficiencies after 100 days of operation were observed with the supplement of carbon substrates. Addition of methanogens inhibitors could effectively inhibit the growth of methanogens with a limited accumulation of TCE dechlorination byproducts [total dichloroethenes and vinyl chloride (VC)] and a higher end-product (ethene) production rate. This indicates that the methanogens growth inhibition resulted in the enhanced TCE dechlorination efficiency. Addition of Mo plus BES caused the highest DHC growth rate (increased from 5 × 103 to 3 × 106 gene copies/L) and TCE dechlorination efficiency (99.8%) after 100 days of incubation. Addition of Clostridium butyricum resulted in the increased hydrogen production and DHC populations, which also improved TCE dechlorination efficiency. Results would provide an effective bioremedial design to cleanup TCE-polluted groundwater.