Abstract Soils were sampled from plots with four variants of tillage methods: (1) conventional tillage (CT); (2) no tillage (NT); (3) minimum tillage (MTS); and (4) no tillage + mulch (NTM). Our aim was to study the influence of tillage on selected soil microbial properties. Determination of urease, dehydrogenase, invertase, arylsulphatase, potential nitrogenase activity, CFU of Azotobacter spp., and carbon microbial biomass has been conducted for time period 2002–2007. Soil samples from 0–0.1 m, 0.1–0.3 m, and 0.3–0.5 m were collected in the spring and autumn. Enzymatic activities (dehydrogenase, urease, arylsulphatase, and invertase) were significantly affected by soil depth and the tillage system employed. The statistically significant higher activity of urease was measured using the minimum tillage system (MTS), compared to the conventional tillage (CT) at soil depths of 0–0.1 m. The highest dehydrogenase activity was measured during the protective tillage treatment (NTM – no tillage with mulch). As far as other enzymatic activities (invertase and arylsulphatase) are concerned, the highest values were recorded in the protective tillage treatments. The highest counts of Azotobacter spp., as well as the highest nitrogenase activity (both statistically significant) were found in the no tillage + mulch (NTM) variant, at depths of 0.1–0.3 m. Microbial biomass (C-biomass) was the highest with the minimum tillage (MTS). The results show a positive influence of protection soil tillage on the reviving of upper layer of topsoil, especially in the variants where soil was supplied with organic matter.