This study investigated the effect of different doses of fertilization with biomass combustion ash (Salix viminalis L. willow) on changes in the biological, chemical, and physical properties of soil. The experiment was carried out on podzolic and chernozem soils in a one-way field experiment (fertilization dose: control (without fertilization), NPK (nitrogen (N), phosphorus (P) and potassium (K)), 100, 200, 300, 400, 500 kg K2O ha−1). The biomass ash was characterized by a pH value of 12.83 ± 0.68 and a high content of macronutrients. The samples were collected from 0–5, 10–15, and 20–25 cm soil layers under the cultivation of spring barley (Hordeum vulgare L) cv. Planet in April and August 2021. Mass spectrometry (MALDI-TOF MS) was used for microbiological analyses, which revealed the presence of 53 culturable species from 11 genera: Bacillus, Pseudomonas, Paenibacillus, Lysinibacillus, Pseudarthrobacter, Arthrobacter, Staphylococcus, Paenarthrobacter, Micrococcus, Rhodococcus, and Flavobacterium. The podzolic and chernozem soils exhibited the presence of 28 and 44 culturable species, respectively. The study showed an increase in the number of microorganisms in the top layer of the soil profile. However, the number of bacteria decreased at the depths of 10–15 cm and 20–25 cm. With depth, the bulk density (BD) and moisture increased.