Freeze-thaw (FT) events exert a great physiological stress on soil microorganisms and hence impact biogeochemical processes in soils. As numerous environmental factors affect microbial and chemical responses to FT, a better understanding of the leverage factors that regulate the responses to FT events is required. To date, FT-induced shifts and transformations in microbial and resource stoichiometry have received particularly little attention. We exposed fifteen Luvisol soils with different time after restoration and corresponding differences in soil organic C contents from a postmining agricultural chronosequence to a single FT event and analysed changes in soil chemistry and microbial stoichiometry one hour and eighteen hours after thawing. FT considerably altered soil biochemical attributes within the first hours of thawing. Microbial biomass C declined substantially after FT, and its relative losses were positively correlated with enhanced dissolved organic C contents. Thus, microbial cell lysis likely led to the significant increase of dissolved organic C. Moreover, microbial biomass C losses were disproportionally higher in C-rich soils, suggesting that soil microorganisms in high-C soils might be particularly prone to FT stress. Microbial biomass N marginally decreased one hour after thawing, yet returned to initial levels eighteen hours after thawing. The alternating responses of microbial biomass C and N caused a strong stoichiometric reduction of the microbial C:N ratio. The resulting microbial oversaturation with N relative to C is likely the first step in the chain of processes that generally lead to the high N losses commonly recorded in agricultural soils in the aftermath of FT events. Metabolic activity of the soil microbial community increased with the relative decline of the microbial biomass C:N ratio eighteen hours after thawing, suggesting increased levels of microbial metabolic expenditure due to stoichiometric shifts. The strength of the FT-driven biochemical responses was strongly dependent on soil organic C content, indicating that high-C soils might be especially vulnerable to initial C and N losses due to shifts in microbial stoichiometry.