Elevated ozone (eO3) and atmospheric nitrogen (N) deposition are important climate change components that can affect plant growth and plant-soil-microbe interactions. However, the understanding of how eO3 and its interaction with N deposition affect soil microbially mediated carbon (C) cycling and the fate of soil C stocks is limited. This study aimed to test how eO3 and N deposition affected soil microbial metrics (i.e., respiration, enzyme activities, biomass, necromass, and community composition) and resulting soil organic C (SOC) fractions in the rhizosphere of poplar plantations with different sensitivity to O3. Exposure to O3 and/or N deposition for four years was conducted within a free-air O3 concentration-enrichment facility. Elevated O3 reduced soil microbial respiration and biomass C but enhanced the enzymatic acquisition of C (i.e., potential soil hydrolase and oxidase activity) and shifted to a fungi-dominated community composition. These responses suggest that microbial C availability decreased and microbes allocated more energy to obtain C and nutrients from biochemically resistant substrates under eO3. Elevated O3 decreased bacterial necromass C and total necromass C, which could explain the observed decreases in mineral-associated organic C and SOC. The effects of eO3 on soil microbial C availability and community composition were strengthened by N addition, whereas there were no differences in the below-ground effects of eO3 between the two poplar clones. Taken together, the increased soil extracellular enzyme activities and slightly increased particulate organic C content suggest that the microbial C pump pathway via microbial ex vivo modification was strengthened by eO3, whereas the pathway via microbial in vivo turnover was weakened, as suggested by the decreases in soil microbial respiration, biomass, necromass, and mineral-associated organic C. Our study provides evidence that aboveground eO3 effects on trees may affect belowground microbial processing of organic matter and ultimately the persistence of SOC.