The relationship between the concentration of soil DNA and microbial biomass, determined by the method of substrate-induced respiration was studied in a wide range of soils differing in particle size distribution, acidity, organic carbon content, microbial biomass, and type of human activity in antiquity and present time. Calcaric Leptosols and Leptic Phaeozems of medieval agricultural lands in the Central Caucasus, Stratozems of agricultural terraces of the late Middle Ages – Modern times in the middle mountain zone of the Eastern Caucasus, as well as Kashtanozems and Solonetzes with different grazing intensity in the dry steppe zone (Rostov region) were chosen as the key sites. It has been shown that the determination of soil dsDNA concentration is a reliable and simple method for determining microbial biomass in soils with a loam texture, organic carbon content less 2%, and microbial biomass less 700 µg C/g of dry soil. The conversion factor FDNA in such soils varied in a narrow range from 5.24 to 5.41. In soils with a high content of organic carbon, an increase of FDNA (6.56 and 10.56) was observed due to the presence of recalcitrant e-xtracellular DNA. Agristratified soil of sandy loam texture was characterized by a lower degree of preservation of dsDNA, which resulted in a decrease in the determined microbial biomass (FDNA = 4.22). A reduced conversion factor FDNA (4.78) was also found in the soils of pastures in the dry steppe zone, which confirms the known limitations of using the substrate-induced respiration method in alkaline soils. Human activity does not significantly affect the relationship between the amount of soil DNA and microbial biomass.