Although the effect of different mycorrhizal types on the soil carbon (C) and nitrogen (N) cycles has been studied intensively, the mismatch between the C:N stoichiometry of microorganisms and their resources (i.e., C:N imbalance) and its impact on microbial physiology remain unclear. In this study, we measured the C:N imbalance, microbial C use efficiency (CUE), and C- and N-acquisition enzyme activities in four tree plantations associated with ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi in subtropical China. We found that the C:N imbalance in ECM plantations was 44% higher than that in AM plantations, which was associated with the lower microbial CUE and higher specific C- and N-acquisition enzyme activities. Collectively, from a novel perspective of ecological stoichiometry, our results demonstrate that the C:N imbalance and the microbial physiological activities it mediated differ between ECM- and AM plantations which have important knock-on consequences for soil C sequestration in the subtropical forests.