Climate change has an impact on agricultural production, including the greater persistence of plant pathogens in the environment. Therefore, the question arises as to how to effectively and safely protect plants by using chemicals, the number of which is decreasing each year. The aim of this study was to evaluate the fungistatic effect of 2 acetylthiophenethiosemicarbazone (2actpTS) and Cis-jasmonethiosemicarbazone (JTS) against Fusarium culmorum (Fc) on winter wheat seedlings. The influence of thiosemicarbazones (TSs) on the health status and phytotoxicity of seedlings and soil biological activity was investigated. Before TSs application (watering or spraying), soil was infected with F. culmorum (strain No. 37). The substance type and method of its application significantly influenced only the fresh weight of the seedlings. The varying phytotoxicity of the molecules depended primarily on their application method to the plants. The highest seedling phytotoxicity was recorded when compounds were applied during watering and the lowest when they were sprayed. The results showed that the application of substances 2actpTS and JTS, both in the form of watering and spraying, had a positive effect on plant health status, as expressed by the disease index (DI) value. The presence of the infectious agent and the type of chemical compound increased soil enzyme activity. The highest total number of bacteria was found in the soil that was infected with Fc and in soil samples where JTS was applied by watering and spraying. The highest utilization of amines, amides and amino acids by microorganisms was found in the samples where JTS was applied. The obtained results will be used to create intermediate products for the protection of cereals against Fusarium diseases, not only at the stage of germination and tillage of plants, but also at subsequent developmental stages.