A biotechnological evaluation was conducted on strains of the genus Trichoderma as key components of biodestructors and soil-improving preparations in the Ecostern® range. Their cultural and morphological characteristics, antagonistic activity against phytopathogenic microfungi, as well as the stability and antifungal efficacy of the Ecostern® Trichoderma biological preparation (gel form) during storage for 3 months and its effect on the structure of the soil microbiota were investigated. The ability of Trichoderma strains to effectively inhibit the growth of Cladosporium herbarum, Botrytis cinerea, Alternaria alternata, Drechslera sorokiniana, Fusarium spp., Rhizoctonia solani and Sclerotinia sclerotiorum was established. The highest antifungal activity was recorded on potato-dextrose agar and a medium containing barley malt extract, where the strains T. harzianum 11/16 TM demonstrated the maximum antagonism index (30–31 points), while T. harzianum 23/1 and T. viride 44/13 LM exhibited high levels of activity (27–28 and 28.5 points, respectively). On Chapek’s medium, antifungal activity decreased by an average of 2.2-fold. The strain of BTU Biotechnology Company T. harzianum surpassed its foreign counterpart in biocontrol potential, demonstrating pronounced mycoparasitism and inhibition of sclerotium formation in S. sclerotiorum. After 3 months of storage, the biopreparation was characterized by a stable titre (1.3–3.0×10⁷ CFU/cm³) and high antifungal activity (79.9–100%) regardless of dilution. A positive effect on the soil mycobiota was also established, with a 5.3-fold reduction in pathogen numbers and an increase in the proportion of saprotrophic fungi to 97.9%. A comprehensive assessment of the antifungal activity and synergistic interaction of microbial strains of the genus Trichoderma enables the identification of patterns governing the development of their biological activity and viability. The results obtained provide a theoretical and practical basis for the development of optimally balanced biological products with high efficacy against plant pathogens.