Despite the potential of subsoil carbon (C) to buffer or amplify climate change impacts, how fresh C and nutrients interact to control microorganismal effects on the C balance in deep soil horizons has yet to be determined. In this study, we aimed to estimate the impact of fresh C input at different soil depths on soil microbial activity. To conduct this study, Mediterranean soils from 3 layers (0–20, 20–50 and 50–100 cm of depth) were incubated over 28 days. Carbon and nutrient fluxes were measured after the addition of an amount of C equivalent to the postharvest root litter derived-C of a barley crop (4.3 atom% 13C), with and without nitrogen and phosphorus supply. We found that the microbial mineralization was C limited in the topsoil, while C and N colimited in the subsoil. These variations in stoichiometric constraints along the soil profile induced different microbial responses to C and/or nutrient addition. A stronger priming effect was observed in the topsoil than in the subsoil, and the sole C addition induced a negative C balance. Conversely, subsoil showed a positive C balance following fresh C addition, changing to critical soil C losses when nutrients were supplied with C. Our results show that fresh C input to subsoil (e.g., through deep-rooting crops) might foster soil C sequestration, but this positive effect can be reversed if such C inputs are combined with high nutrient availability (e.g., through fertilization), alleviating microbial limitation at depth.