The research was carried out on the territory of the Russian Federation in the forest-steppe region of the south Western Siberia (Omsk state), in the long-term (43 years) stationary experiment. Sprinkling was used for irrigation in the experiment. The number of different physiological groups of microorganisms, the cellulolytic activity of the soil, and nitrification capacity were determined under the sowing of an eight-field grain-grass crop rotation (perennial grasses (Bunias orientalis L. + Bromopsis inermis L. + Galega orientalis Lam. 6–8 years old), spring barley Hordeum vulgare Leyss.—variety Sasha). Immobilization processes predominated in the soil under the sowed crops, it contributes to the preservation of soil organic matter (mineralization coefficient SAA/MPA < 1). The highest transformation ratio of soil organic matter, i.e. increased conversion of plant residues into organic matter, was noted with applying nitrogen-phosphorus fertilizers (N60P60) under the barley. The combination of irrigation factors and the use of mineral fertilizers (N30-60P60) were contributed to the growth of the microorganisms’ population, the amplification of decomposition of cellulose, and improvement of nitrification capacity in the soil. The perennial irrigation of the meadow-chernozem soil and the application of intensive technology of cultivation of crops in crop rotation stimulated the growth of the microorganisms’ population and didn’t detriment the ecological state of the soil.