Chloroacetamide herbicides are widely used to control weeds globally. In this study, three acetochlor-degrading mixed cultures using nitrate, sulfate, and ferric iron as electron acceptors were isolated and determined for their degradation under anaerobic conditions. The degradation rates of all mixed pure cultures in a mineral medium were not much different at 1 µM, while the rates at 50 µM were in the order: mixed culture using nitrate > sulfate > ferric iron as electron acceptors, giving 6.70, 6.13, and 4.85 µM/day, respectively. During acetochlor degradation, 2-ethyl-6-methyl-N-(ethoxymethyl)acetanilide, N-2-ethylphenyl acetamide, and 2-ethylaniline were transiently produced. Interestingly, the mixture of all cultures synergistically degraded pretilachlor although no individual strains could degrade the compound. Toluene and 2-ethylaniline were metabolites of pretilachlor degradation by the acetochlor-degrading bacteria. However, pretilachlor inhibited the acetochlor degradation by acetochlor-degrading bacteria. The inoculation of both acetochlor- and pretilachlor-degrading bacteria resulted in 91.4±2.1% of acetochlor and 82.4±2.6% of pretilachlor being utilized for 7 days. Moreover, the introduction of these degrading bacteria accelerated the degradation of both substrates contaminating water and sediment collected from a pond. This study provides insights into anaerobic degradation by pure cultures using different electron acceptors.