Soil microbial and enzyme activities are closely related to the spatial variability of soil environmental conditions at the microscale (μm-mm). The origin and localization of the enzymes are somewhat neglected when the measured activity is used to evaluate specific soil functions. The activity of four hydrolytic enzymes (β-glucosidase, Cellobiohydrolase, Chitinase, Xylanase) and microbial diversity based on community-level physiological profiling were determined in samples of arable and native Phaeozems with increasing physical impact to soil solids. The level of impact on the soil solids had a significant effect on enzyme activity and depended on both the enzyme type and soil land use. The highest proportion of the activity of Xylanase and Cellobiohydrolase of arable Phaeozem was determined at the dispersion energy in the range of 450–650 J·mL−1and was associated with the primary soil particles’ hierarchy level. The highest proportions ofβ-glucosidase and Chitinase activities were determined for forest Phaeozem after applying energies lower than 150 J·mL−1and characterizing the level of soil microaggregates. The increased activity of Xylanase and Cellobiohydrolase in primary soil particles of arable soil compared to those in forest soil might be a reflection of the substrates being unavailable to decomposition, leading to enzyme accumulation on the solid surface. For the Phaeozems, the lower the level of soil microstructure organization, the greater the differences observed between soils of different land use type, i.e., microbial communities, associated with lower microstructure levels, were more specific to land use type.