Phenoxyalkanoic herbicides such as 2,4-dichlorophenoxyacetate (2,4-D), 2,4-dichlorophenoxybutyrate (2,4-DB) or mecoprop are widely used to control broad-leaf weeds. Several bacteria have been reported to degrade these herbicides using the α-ketoglutarate-dependent, 2,4-dichlorophenoxyacetate dioxygenase encoded by the tfdA gene, as the enzyme catalysing the first step in the catabolic pathway. The effects of exposure to different phenoxyalkanoic herbicides in the soil bacterial community and in the tfdA genes diversity were assessed using an agricultural soil exposed to these anthropogenic compounds. Total community bacterial DNA was analysed by terminal restriction fragment length polymorphism of the 16S rRNA and the tfdA gene markers, and detection and cloning of tfdA gene related sequences, using PCR primer pairs. After up to 4 months of herbicide exposure, significant changes in the bacterial community structure were detected in soil microcosms treated with mecoprop, 2,4-DB and a mixture of both plus 2,4-D. An impressive variety of novel tfdA gene related sequences were found in these soil microcosms, which cluster in new tfdA gene related sequence groups, unequally abundant depending on the specific herbicide used in soil treatment. Structural analysis of the putative protein products showed small but significant amino acid differences. These tfdA gene sequence variants are, probably, required for degradation of natural substrate(s) structurally related to these herbicides and their presence explains self-remediation of soils exposed to phenoxyalkanoic herbicides.