Climate change factors such as elevated atmospheric carbon dioxide (CO₂) and ozone (O₃) can exert significant impacts on soil microbes and the ecosystem level processes they mediate. However, the underlying mechanisms by which soil microbes respond to these environmental changes remain poorly understood. The prevailing hypothesis, which states that CO₂- or O₃-induced changes in carbon (C) availability dominate microbial responses, is primarily based on results from nitrogen (N)-limiting forests and grasslands. It remains largely unexplored how soil microbes respond to elevated CO₂ and O₃ in N-rich or N-aggrading systems, which severely hinders our ability to predict the long-term soil C dynamics in agroecosystems. Using a long-term field study conducted in a no-till wheat-soybean rotation system with open-top chambers, we showed that elevated CO₂ but not O₃ had a potent influence on soil microbes. Elevated CO₂(1.5×ambient) significantly increased, while O₃ (1.4×ambient) reduced, aboveground (and presumably belowground) plant residue C and N inputs to soil. However, only elevated CO₂ significantly affected soil microbial biomass, activities (namely heterotrophic respiration) and community composition. The enhancement of microbial biomass and activities by elevated CO₂ largely occurred in the third and fourth years of the experiment and coincided with increased soil N availability, likely due to CO₂-stimulation of symbiotic N₂ fixation in soybean. Fungal biomass and the fungi∶bacteria ratio decreased under both ambient and elevated CO₂ by the third year and also coincided with increased soil N availability; but they were significantly higher under elevated than ambient CO₂. These results suggest that more attention should be directed towards assessing the impact of N availability on microbial activities and decomposition in projections of soil organic C balance in N-rich systems under future CO₂ scenarios.