The decomposition of soil organic matter is mediated by extracellular enzymes. The aim of this work was to identify the factors determining the activity and size of the mobile fraction of extracellular enzymes (laccase, Mn-peroxidase, endocellulase, cellobiohydrolase, β-glucosidase, endoxylanase, β-xylosidase, α-glucosidase, chitinase, arylsulfatase, phosphatase, phosphodiesterase, alanine and leucine aminopeptidase) using a set of soils covering a wide range of physico-chemical properties. Organic matter content had a major effect on enzyme activity both in forest and grassland soils, while the effects of pH and humic compounds content were only important in forest soils, and the molecular mass of humic compounds and Ca content were only important in grasslands. Specific enzyme activity was either comparable between the soil types or higher in grasslands. With the exception of Mn-peroxidase and β-glucosidase, the specific activities of all enzymes in arable fields under tillage were similar to those in grasslands. Mobility differed among the enzymes and ranged from <1% for arylsulfatase and phosphodiesterase up to 20–40% for α-glucosidase and aminopeptidases, with pH being the most important variable. These results demonstrate that the factors regulating enzyme activity are likely to be different in forest soils and grasslands and that enzyme mobility is a characteristic feature of each individual enzyme.