Soil microorganisms are limited by the amount and type of plant-derived substrates entering soil, and we reasoned that changes in the production and biochemical constituents of plant litter produced under elevated CO2 and O3 would elicit physiological changes in soil microbial communities. To test this idea, we studied microbial activity beneath trembling aspen (Populus tremuloides Michx.), paper birch (Betula papyrifera Marsh.), and sugar maple (Acer saccharum Marsh.) growing under experimental atmospheric CO2 (ambient and 522.7 μL L-1) and O3 (ambient and 54.5 nL L-1). To assess changes in microbial community function, we measured microbial biomass, respiration, and the metabolism of root-derived substrates using BIOLOG GN microplates. We also measured the activity of phosphatase, leucine aminopeptidase, α-glucosidase, N-acetylglucosaminidase, cellobiohydrolase, phenol oxidase, and peroxidase enzymes, which are involved in plant and fungal litter decomposition. Microbial biomass and respiration were not significantly altered by elevated CO2 and O3. Cellobiohydrolase activity significantly increased under elevated CO2; however, this response was eliminated by elevated O3. N-acetylglucosaminidase activity also increased under elevated CO2, but elevated O3 did not significantly alter this response. We found no difference in the metabolism of amino acids, organic acids, and simple carbohydrates, suggesting our experimental treatments did not alter the use of these substrates by soil microorganisms. Our analysis indicates that changes in plant growth in response to elevated CO2 and O3 alters microbial metabolism in soil.