Fungicides are considered to be effective crop protection chemicals in modern agriculture. However, they can also exert toxic effects on non-target organisms, including soil-dwelling microbes. Therefore, the environmental fate of fungicides has to be closely monitored. The aim of this study was to evaluate the influence of the Falcon 460 EC fungicide on microbial diversity, enzyme activity and resistance, and plant growth. Samples of sandy loam with pH KCl 7.0 were collected for laboratory analyses on experimental days 30, 60 and 90. Falcon 460 EC was applied to soil in the following doses: control (soil without the fungicide), dose recommended by the manufacturer, 30-fold higher than the recommended dose, 150-fold higher than the recommended dose and 300-fold higher than the recommended dose. The observed differences in the values of the colony development index and the eco-physiological index indicate that the mixture of spiroxamine, tebuconazole and triadimenol modified the biological diversity of the analyzed groups of soil microorganisms. Bacteria of the genus Bacillus and fungi of the genera Penicillium and Rhizopus were isolated from fungicide-contaminated soil. The tested fungicide inhibited the activity of dehydrogenases, catalase, urease, acid phosphatase and alkaline phosphatase. The greatest changes were induced by the highest fungicide dose 300-fold higher than the recommended dose. Dehydrogenases were most resistant to soil contamination. The Phytotoxkit test revealed that the analyzed fungicide inhibits seed germination capacity and root elongation. The results of this study indicate that excessive doses of the Falcon 460 EC fungicide 30-fold higher than the recommended dose to 300-fold higher than the recommended dose) can induce changes in the biological activity of soil. The analyzed microbiological and biochemical parameters are reliable indicators of the fungicide's toxic effects on soil quality.