Although there is evidence that cover crops enhance soil microbial diversity and activity, limited information is available about the impact of cover crops on biological indicators of soil health. The use of multi-species cover crop mixtures has been promoted nationwide to enhance soil health; however, little data are available demonstrating the benefits of cover crop mixtures compared with a single species in the Southeastern U.S. This project examined the effects of three winter cover crop species grown singly and combined in multispecies mixtures on selected biological indicators of soil health and determined the changes of these indicators over a three-year period.