The aim of this research was to determine if short and/or long term exposure to 2,4-dichlorophenoxyacetic acid (2,4-D) impacts the structure and function of microbial communities of agricultural soils. We studied short-term effects in agricultural soil microcosms spiked with different levels of 2,4-D (0, 5, and 50 mg/kg) by assessing C sources utilization profiles using 96-well BD^TM Oxygen Biosensor System (BD-Oxy) plates up to 20 days after treatment. The BD-Oxy assay measures community respiration via the fluorescence of an O₂-quenched fluorophore; substrates tested included casein, acetate, sucrose, mannose, succinate, coumaric acid, 2.4-D and no substrate amendment. In general, substrates utilization was not significantly changed by 2,4-D treatment, although succinate, casein and acetate utilization was higher in treated microcosms at days 7 and 14, respectively. Microcosms treated with 50 mg/kg of 2,4-D exhibited a greater response to 2,4-D as substrate than microcosms treated with 5 mg/kg. The substrate utilization results indicate that agriculturally relevant levels of 2,4-D leads to selection of 2,4-D degraders, but has little effect on overall heterotrophic activity. Long-term effects were evaluated by assessing pollution-induced community-tolerance (PICT) to the herbicide. The PICT method was performed by evaluating the respiratory response to coumaric acid in agricultural soils currently receiving 2,4-D or not (and a reference, pristine forest soil) in the presence of increasing concentrations of 2,4-D. The PICT assay found that both treated and untreated microcosms were highly tolerant of 2,4-D, but that the reference forest soil was relatively intolerant. The agricultural soil used in this study, although not from a site with active 2,4-D use, contained a community tolerant to 2,4-D exposure, suggesting that either previous exposure to 2,4-D led to a persistently tolerance community, or that the agricultural soils are more generally resistant to potential stressors.