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Global soil carbon (C) stocks are expected to decline with warming, and changes in microbial processes are key to this projection. However, warming responses of critical microbial parameters such as carbon use efficiency (CUE) and biomass turnover (rB) are not well understood. Here, we determine these parameters using a probabilistic inversion approach that integrates a microbial-enzyme model with 22 years of carbon cycling measurements at Harvard Forest. We find that increasing temperature reduces CUE but increases rB, and that two decades of soil warming increases the temperature sensitivities of CUE and rB. These temperature sensitivities, which are derived from decades-long field observations, contrast with values obtained from short-term laboratory experiments. We also show that long-term soil C flux and pool changes in response to warming are more dependent on the temperature sensitivity of CUE than that of rB. Using the inversion-derived parameters, we project that chronic soil warming at Harvard Forest over six decades will result in soil C gain of <1.0% on average (1st and 3rd quartiles: 3.0% loss and 10.5% gain) in the surface mineral horizon. Our results demonstrate that estimates of temperature sensitivity of microbial CUE and rB can be obtained and evaluated rigorously by integrating multidecadal datasets. This approach can potentially be applied in broader spatiotemporal scales to improve long-term projections of soil C feedbacks to climate warming.