Many agriculturalists have been focusing on the most efficient farming method that would produce the maximum yield while still sustaining the soil ecosystem. Soil samples were collected from the “ Old Rotation ” area (Auburn University, Auburn, AL ), and were assessed for soil biochemical, chemical and biological characteristics related to soil quality. Treatments of the experimental site were a control with no legumes or N fertilizer; cotton every year with winter legumes ; a 3 - yr cotton - corn - soybean rotation with wheat and winter legumes ; and cotton every year with N fertilizer . Impacts of irrigation were also tested between the sites. Assays were performed measuring phosphomonoesterase and phosphodiesterase activity, soil organic carbon , soil pH , and microbial diversity. The 3 - year and winter legume rotation s showed significant differences in the structure and membership of microbial communities and differences in biochemical activity. These results further demonstrate the ability of crop rotation to enhance the soil health of agricultural ecosystems.