Crop varieties resistant to multiple herbicides have been developed to provide better control of weed populations in row-crop fields where glyphosate resistance has become common. These new varieties include lines of maize (Zea mays) resistant to both glyphosate and 2,4-dichlorophenoxyacetic acid (2,4-D). As these herbicides have the potential to impact microbial communities in soil, there is concern that their co-application may have a greater effect on activities linked to soil nutrient cycling than if they were applied individually. To investigate this possibility, a field study was conducted on 2,4-D+glyphosate-resistant maize to determine the impact of 2,4-D alone and 2,4-D+glyphosate on extracellular enzyme activity in both bulk and rhizosphere soil. Maize was treated at the V2 and V8 developmental stages. Changes in soil activities were small in magnitude and inconsistent between timepoints. 2,4-D+glyphosate-treated plots had higher beta-glucosidase, cellobiohydrolase, and phosphatase activities, but only after the V2 application in bulk soil in the first year of the study, while no significant effects were observed in the rhizosphere. Enzyme activities were more impacted by soil organic matter than herbicide treatments. These results suggest that, when applied at label rates, 2,4-D+glyphosate application will not adversely affect soil microbial enzyme activities.