Determination of the microbial and enzymatic properties in soil is primarily concentrated on the surface layers of the soil profiles; however, it is well known that the transformation of soil organic matter also occurs in the deeper horizons of the soil profile. The aim of this study was to assess any changes in specific sets of enzyme activities and their associated physicochemical properties as affected by two different agricultural land-use systems and soil depth. Changes in the studied properties were determined across four Luvisol profiles in two agricultural land uses (arable land and vineyards). The enzyme activities associated with the transformation of C, N and P were analyzed. Additionally, the activity of some oxidoreductases and the fluorescein diacetate hydrolysis (FDAH) rate were also determined. Moreover, the content of the various forms of soil carbon, nitrogen, phosphorus (including microbial biomass C, N and P) and some other properties (pH, clay and silt content) were assessed. Agricultural land use significantly affected the microbial biomass content and as well as the studied enzyme activities. Most of the studied enzymes exhibited a higher activity in the grapevine (GV) profiles, which was followed by the winter wheat (WW) profiles; however, the largest variability occurred for the urease activity. There was no clear differentiation between the two studied land uses for the activity of nitrate reductase, dehydrogenases, acid phosphatase, or endo- and exo-cellulase. Irrespective of the plant being cultivated, the soil variables decreased significantly with increasing soil depth, wherein the greatest changes were observed between the surface and sub-surface soil horizons (I–II). The activity of some enzymes (e.g., the urease activity in WW profiles) decreased gradually across the soil profiles, while others were located almost solely within the surface layers (e.g., the nitrate reductase activity in the GV profiles as well as invertase in the WW profiles). The α-glucosidase activity did not exhibit any statistically significant changes along the analyzed profiles. The activity of phenol oxidase and peroxidase also revealed different trends along the studied profiles compared to the other enzymes and did not decrease gradually with depth. The microbial biomass of the C, N and P content was generally the highest in the upper horizons and gradually decreased with depth, wherein the largest decrease was observed between the surface and sub-surface horizon. The studied enzyme activities were more dependent on the soil carbon content compared to the other soil properties. And thus, in the C-rich horizons (C > 4 g kg) for the surface and subsurface layers the enzyme activities were highly correlated with TOC, DOC and MBC content as compared to the deeper, C-low horizons (C < 4 g kg). By examining how the microbial and enzymatic properties change across the soil profiles, it is possible to gain valuable insight into the long-term biogeochemical processes that are involved in soil fertility and in the health of agricultural ecosystems.