Pesticide contamination is one of the most serious threats for agricultural soils. Excessive pesticide levels in soil can exert negative effects on soil-dwelling organisms by decreasing their bioavailability and, consequently, lowering soil quality. This study aimed to evaluate the effect of a mixture of spiroxamine, tebuconazole, and triadimenol (S + Te + Tr) on the biological activity of soil determined based on the proliferation of microorganisms and their diversity, enzymatic activity of soil, and resistance of Triticum aestivum L. A pot experiment was performed on sandy loam (pH 7.0) treated with four doses of the tested active ingredients: 0.000, 0.092, 2.76, 13.80, and 27.60 mg kg. Soil without the fungicide served as the control sample. The tested fungicide induced changes in the biological activity of soil. When administered to the soil in the highest dose (27.60 mg kg DM of soil), it inhibited its biological activity. It significantly inhibited the proliferation of organotrophs, actinomycetes, and fungi, but still the most susceptible to its effects turned out to be fungi. Fungicide dose of 27.60 mg kg decreased counts of organotrophic bacteria, actinomycetes, and fungi by on average 0.009 log, 0.100 log, and 0.282 log, respectively, compared to the control sample. Administration of the S + Te + Tr mixture to soil decreased also values of colony development index (CD) determined for all tested groups of microorganisms. Values of the ecophysiological diversity index (EP) decreased in the case of organotrophs and actimomycetes and increased in the case of fungi. The S + Te + Tr mixture inhibited activities of dehydrogenases, urease, and acid phosphatase. Significant changes were also reported in the growth of spring wheat. The resistance index (RS) calculated based on plant yield demonstrated spring wheat to be very susceptible to the tested preparation administered to soil in doses of 13.80 and 27.60 mg kg.