Medicinal plants are one of the groups of economically important species and a valuable natural resource. One of these is thyme (Thymus vulgaris L.), the source of essential oil with high antimicrobial activity against microorganisms from various systematic groups. The study of thyme–microbial interactions was carried out in 2014-2024 for the collection of medicinal plants of the V. I. Edelstein Vegetable Experimental Station by cultural, chromatography, and NGS methods. The data obtained indicated the importance of the phytosphere's physiological and biochemical features for microbial community structure and representation depending on the development stage, since these factors significantly affected the plant–microbial interaction in the thyme–soil system. In other words, the features of the studied plant–microbiological interaction depended on which microorganisms inhabited specific parts of the thyme phytosphere and at what stage of plant development this occurred. Moreover, for bacterial communities, the key roles were played by the features of the phenological stage of the plant (54.3%) and the colonized part (44.7%), while for fungi, the interaction of these two factors was the most significant (75.8%). For microbial biotechnologies, it is necessary to obtain cultured identified forms of microorganisms. So, typical representatives of microbial communities were isolated and sequenced. At least nine phytosphere microorganisms can be recommended as protective and growth-regulating agents for thyme cultivation in the Non-Chernozem zone due to the suppression of root rot pathogens that actively develop during thaws and high humidity conditions.