The content of microbial biomass (MB) was determined in samples of gray forest, chestnut, and tundra soils with different physicochemical properties (0.4–22.7% Corg; 8.4–26.8% silt particles; pH 4.3–8.4) by the methods of substrate-induced respiration (MBSIR) and direct microscopy (MBM). The samples of two upper soil layers, 0–5 and 5–10 cm (without plant litter), from different ecosystems (forest, forest shelter belt, meadow, fallow, and arable) and elements of relief of interfluvial tundra (block/upper land plateau, depression between blocks) have been analyzed. The content of microbial biomass in the 0–5-cm soil layer was 216–8134 and 348–7513 μg C/g soil as measured by the methods of substrate-induced respiration and direct microscopy, respectively. The MBSIR and MBM values closely correlated with each other: r = 0.90 and 0.74 for 0–5 and 5–10 cm, respectively. The average MBSIR/MBM ratio was 90 and 60% for 0–5 and 5–10 cm, respectively. The portion of microbial carbon in total organic soil carbon was, on average, 4 and 3% (SIR) and 5 and 7% (direct microscopy) for 0–5 and 5–10 cm, respectively. Possible reasons for the differences between MBSIR and MBM values in the soils under study are discussed.