Tillage is the common practice in crop production, providing a suitable environment for root development. However, many studies suggest that long-term tillage practice affects the deterioration of soil properties. Soil biological indicators can be employed to evaluate the soil changing due to the long-term tillage application. The evaluation of soil biological indicators in the long-term tillage application has not been widely discussed in Hungary. We investigated two types of 19 years of long-term tillage experiments, i.e., conservation tillage, reduced tillage and leaving 30% or more crop residue after harvesting (CT), and conventional tillage, a mouldboard ploughing technique (PT). The soil was sampled thrice during the growing season at 0-5, 10-15, and 20-25 cm depths. The activity of dehydrogenase (DHA), β-glucosidase (BGA), phosphatase (PHA), labile carbon concentration (POXC), and soil water content (SWC) were measured in this investigation. Our results indicated that the tillage system significantly affected the BGA (p<0.001). Meanwhile, soil depth remarkably regulated the DHA (p<0.05). The highest activity of DHA and BGA was identified near the surface layer of CT. In contrast, the PHA was relatively distributed uniformly in all depths, reflecting that this enzyme activity was more dominated by the root effect. Tillage practice notably increases substrate availability, representing the higher POXC concentration in the CT and PT in all soil layers. POXC concurrent with SWC were remarkably associated with DHA (r=0.51, p<0.01), BGA (r=0.29, p<0.05), and PHA (r=0.28, p<0.05). On the other hand, POXC was notably correlated only with the DHA (r=0.46, p<0.01). CT practice also indicated a remarkably positive effect on stem and flower diameter growth.