Soil microorganisms play a critical role in biogeochemical cycles that affects crop production through nutrient regulation and availability, plant health, soil structure and organic matter dynamics.  This project aimed to characterize the microbial community size, structure and activity as affected by tillage (till and no-till), nitrogen (N) fertilizer rates (0N and 101N kg/ha), and cover crops (Vetch- Vicia villosa and Wheat- Triticum aestivum) for cotton production in long-term plots (31 yrs).  The research was established in 1981 at the West Tennessee Research and Education Center (WTREC), located at Jackson, Tennessee.  Results from PCA indicated that N rate and tillage clearly influenced total C and N, and the microbial community structure according to Fatty Acid Methyl Ester (FAME) profiles. Nitrogen application rates increased microbial respiration (MR) and microbial biomass C (MBC) with treatments under high N having greater MR and MBC. Tillage had a significant influence on the MBC, and total C: total N ratios which were higher in no-till (NT) treatments.  There was a nitrogen by tillage interaction effect on total C and total N, with NT-high N treatments having significantly higher levels of total C and totalN. Treatments under vetch, a nitrogen-fixing cover crop species, had significantly higher total N levels compared to those under wheat or no cover. A combination of NT with vetch cover resulted in a significant increase of TC while treatments having wheat cover crop had the lowest TC.  The relative abundance of bacteria and fungi were also greatly influenced by N rates and tillage. Treatments under high N and NT had a higher relative abundance of bacteria. Conversely, the relative abundance of fungi was lower in high N treatments and also relatively lower under NT treatments. These results suggest that microbial processes are dynamic and impacted by the combined factors of tillage, N rates, cover crop species and merits further research and subsequent analysis.