Abstract The activities of extracellular enzymes that participate in the decomposition of litter and organic matter in forest soils depend on, among other factors, temperature and soil moisture content and also reflect the quality of litter, which changes dramatically after a short litterfall period. Here, we explored the effects of soil temperature and seasonality on the sizes of extracellular enzyme pools and activities in a temperate hardwood forest soil with dominant Quercus petraea (cambisol, mean annual temperature 9.3 °C). We hypothesized that the most significant variation of enzyme activity would occur in the litter, which faces greater variations in temperature, moisture content and chemical quality during the season, which decrease with soil depth. The site exhibited relatively large seasonal temperature differences and moderate changes in soil moisture content. Enzyme activity, microbial biomass, soil moisture content, temperature and pH were monitored for three years in the litter (L), organic horizon (O) and upper mineral horizon (Ah). Enzyme activity in vitro strongly increased with temperature until 20–25 °C, the highest temperatures recorded in situ. While no significant differences in the pools of most extracellular enzymes and in the content of microbial biomass were found among the seasons, enzyme activity typically increased during the warm period of the year, especially in the O and Ah horizons. Approximately 63%, 64%, and 69% of total annual activity was recorded during the warm period of the year in the L, O, and Ah horizons, respectively. Significant positive correlations were observed between soil moisture content and fungal biomass, but not bacterial biomass, indicating a decrease of the fungal/bacterial biomass ratio under dry conditions. The effect of moisture on enzyme activities was not significant except for endoxylanase in the litter. If soil temperature rises as predicted due to global climate change, enzyme activity is predicted to rise substantially in this ecosystem, especially in winter, when decomposition is not limited by drought and fresh litter that can decompose rapidly is present.