Ozone (O3) and nitrogen (N) deposition affect plant carbon (C) dynamics and may change ecosystem C-sink/-source properties. We studied effects of increased background [O3] (up to [ambient] × 2) and increased N deposition (up to +50 kg ha−1 a−1) on mature, subalpine grassland during the third treatment year. During 10 days and 13 nights, distributed evenly over the growth period of 2006, we measured ecosystem-level CO2 exchange using a static cuvette. Light dependency of gross primary production (GPP) and temperature dependency of ecosystem respiration rates (Reco) were established. Soil temperature, soil water content, and solar radiation were monitored. Using Reco and GPP values, we calculated seasonal net ecosystem production (NEP), based on hourly averages of global radiation and soil temperature. Differences in NEP were compared with differences in soil organic C after 5 years of treatment. The high [O3] had no effect on aboveground dry matter productivity (DM), but seasonal mean rates of both Reco and GPP decreased ca. 8%. NEP indicated an unaltered growing season CO2–C balance. High N treatment, with a +31% increase in DM, mean Reco increased ca. 3%, but GPP decreased ca. 4%. Consequently, seasonal NEP yielded a 53.9 g C m−2 (±22.05) C loss compared with control. Independent of treatment, we observed a negative NEP of 146.4 g C m−2 (±15.3). Carbon loss was likely due to a transient management effect, equivalent to a shift from pasture to hay meadow and a drought effect, specific to the 2006 summer climate. We argue that this resulted from strongly intensified soil microbial respiration, following mitigation of nutrient limitation. There was no interaction between O3 and N treatments. Thus, during the 2006 growing season, the subalpine grassland lost >2% of total topsoil organic C as respired CO2, with increased N deposition responsible for one-third of that loss.