The regular application of mineral and organic fertilizers is one of the essential components of the agricultural intensification. The applied fertilizers lead to artificial enrichment of the soil environment with readily available substrates and biophilic elements, which can have a significant impact on the soil and rhizosphere microbiome. The aim of the study was to investigate the effect of different fertilizer systems on the quantitative parameters of various microbial groups in soddy-podzolic soil (Umbric Albic Retisol) and the rhizosphere of potatoes and barley. The study was carried out on a long-term field experiment, in which mineral (NPK), organic (manure) and mixed (NPK + manure) fertilizer systems have been applied since 1968. The application of organic fertilizers increased the microbial biomass carbon (Cmic) in the bulk soil and the rhizosphere by 25–100% compared to the soil without fertilizers, while the use of mineral fertilizers, on the contrary, decreased it by 10–30%. Basal respiration and respiratory coefficient (qСО2) increased in the row: without fertilizers NPK NPK + manure manure. The gene copies number of bacteria, archaea, and fungi significantly increased (1.5–2.5 times) under organic fertilizers and decreased (2–2.5 times) under NPK. The varied from 32 to 100 and from 0.10 to 0.92 by the luminescent microscopy and quantitative PCR, respectively. The lowest fungi/bacteria ratios were revealed for variants with NPK, and the highest fungi/bacteria ratios were in variants with manure. Thus, the applied doses of mineral fertilizers must be compensated by the introduction of fresh organic matter in order to maintain the stability of the soil-microbe-plant system.