It is found that the 70-year application of fertilizers in a grain-sugar beet crop rotation in the forest steppe of the Central Chernozem Region changes considerably the biochemical indices of leached chernozem fertility and increases the activity of urease by 17.4–26.9%, catalase by 5.6–36.5%, protease by 23.2–54.2%, cellulose-decomposing activity by 96.2–154.2%; phosphatase activity decreased by 65.7–105.1%. The phytotoxicity of soil against a high fertilizer dose background increased 85.7%.