Agronomic practices can have a negative impact on soil health and quality and ecosystem resilience. The objectives of the study were (1) to evaluate the soil chemical properties and microbial abundance under short-term application of organic and mineral fertilizers and different cultivated crops and (2) to observe the antifungal efficacy of microorganisms isolated from the studied soil. A field trial was conducted in the 2021–2022 period on a preluvosoil-type soil in four randomized blocks with eight fertilizer treatments based on manure compost (MC) and MC + mineral fertilizer (V1—control-soil; V2—NPK only; V3—15 t/ha MC; V4—15 t/ha MC + NPK; V5—30 t/ha MC; V6—30 t/ha MC + NPK; V7—60 t/ha MC; and V8—60 t/ha MC +NPK) and four crops (winter wheat—Triticum aestivum L., maize—Zea mays L., soybean—Glycine max L., and a mixture of perennial grasses and legumes). In almost all treatments, the soil pH decreased during the summer–autumn period. The organic carbon (Corg) and humus contents increased compared to the initial state of the soil after the application of different doses of MC and MC + NPK fertilization in almost all treatments. The microbial load of the soil was influenced by the fertilization regime and crop species, but there were no significant differences between the variants. The highest bacterial load was recorded in soil cultivated with a mixture of perennial grasses and grain legumes, i.e., in the variant with 15 t/ha MC, followed by soil cultivated with maize and fertilized with 30 t/ha and 60 t/ha MC. A higher number of fungi was observed in the mixture of perennial grasses and legumes, and Rhizobium population was higher, especially in the winter wheat plots, despite the fertilization regime. The antifungal efficacy of the microorganisms isolated from the samples was medium to low, except in the winter wheat experiment, where the efficacy against Fusarium culmorum was medium to high and against other pathogens was medium. In the other crops within the experiment, the antagonistic activity of the soil microorganisms was medium to low.