Abstract2,4-Dichlorophenoxyacetic acid (2,4-D) is the third most applied pesticide in Brazil to control broadleaf weeds in crop cultivation and pastures. Due to 2,4-D’s high mobility and long half-life under anoxic conditions, this herbicide has high probability for groundwater contamination. Bioremediation is an attractive solution for 2,4-D contaminated anoxic environments, but there is limited understanding of anaerobic 2,4-D biodegradation. In this study, methanogenic enrichment cultures were obtained from Amazonian top soil (0—40 cm) and deep soil (50 -80 cm below ground) that biotransform 2,4-D (5 µM) to 4-chlorophenol and phenol. When these cultures were transferred (10% v/v) to fresh medium containing 40 µM or 160 µM 2,4-D, the rate of 2,4-D degradation decreased, and biotransformation did not proceed beyond 4-chlorophenol and 2,4-dichlorophenol in the top and deep soil cultures, respectively. 16S rRNA gene sequencing and qPCR of a selection of microbes revealed no significant enrichment of known organohalide-respiring bacteria. Furthermore, a member of the genus Cryptanaerobacter was identified as possibly responsible for phenol conversion to benzoate in the top soil inoculated culture. Overall, these results demonstrate the effect of 2,4-D concentration on biodegradation and microbial community composition, which are both important factors when developing pesticide bioremediation technologies.