Temperature and substrate availability are among the key factors controlling microbial metabolism. The relative importance of these two drivers on soil organic matter turnover is, however, hotly debated. In this study, we investigated the effect of temperature changes on the potential enzyme activities involved in C (phenol-oxidase) and N (protease and amidase) cycling by incubating peat soils collected in winter and summer at the two typical temperatures recorded in the field during these two distinct periods (4 and 19 °C, respectively). In addition, to evaluate the effect of substrate limitations, we also compared the respiration rates of the thermally adapted soils with and without plant litter additions. Results showed that both collection season and incubation temperature had a significant effect on the two enzymes involved in N-cycling, with summer and increasing temperatures having detrimental effects on the potential activities of protease and amidase, whereas none of these factors affected phenol-oxidase activity. Furthermore, while adding readily decomposable substrate accelerated decomposition rates, CO2 flux rates were similar for all temperature conditions. Interestingly, the greatest contribution of litter to CO2 emissions occurred in the summer samples incubated at the lower temperature, whereas for the winter samples the stimulating effect on soil respiration was observed under warmer conditions. These findings suggest that the responses of soil microbial communities to temperature and substrate availability seem to strongly depend on the long-term temperature conditions and its interaction with substrate availability.