Soil microbial biomass C (Cmic) is a sensitive indicator of trends in organic matter dynamics in terrestrial ecosystems. This study was conducted to determine the effects of tropospheric CO2 or O3 enrichments and moisture variations on total soil organic C (Corg), mineralizable C fraction (CMin), Cmic, maintenance respiratory (qCO2) or Cmic death (qD) quotients, and their relationship with basal respiration (BR) rates and field respiration (FR) fluxes in wheat-soybean agroecosystems. Wheat (Triticum aestivum L.) and soybean (Glycine max. L. Merr) plants were grown to maturity in 3-m dia open-top field chambers and exposed to charcoal-filtered (CF) air at 350 μL CO2 L−1; CF air + 150 μL CO2 L−1; nonfiltered (NF) air + 35 nL O3 L−1; and NF air + 35 nL O3 L−1 + 150 μL CO2 L−1 at optimum (− 0.05 MPa) and restricted soil moisture (− 1.0 ± 0.05 MPa) regimes. The + 150 μL CO2 L−1 additions were 18 h d−1 and the + 35 nL O3 L−1 treatments were 7 h d−1 from April until late October. While Corg did not vary consistently, CMin, Cmic and Cmic fractions increased in soils under tropospheric CO2 enrichment (500 μL CO2 L−1) and decreased under high O3 exposures (55 ± 6 nL O3 L−1 for wheat; 60 ± 5 nL O3 L−1 for soybean) compared to the CF treatments (25 ± 5 nL O3 L−1). The qCO2 or qD quotients of Cmic were also significantly decreased in soils under high CO2 but increased under high O3 exposures compared to the CF control. The BR rates did not vary consistently but they were higher in well-watered soils. The FR fluxes were lower under high O3 exposures compared to soils under the CF control. An increase in Cmic or Cmic fractions and decrease in qCO2 or qD observed under high CO2 treatment suggest that these soils were acting as C sinks whereas, reductions in Cmic or Cmic fractions and increase in qCO2 or qD in soils under elevated tropospheric O3 exposures suggest the soils were serving as a source of CO2.