Crop management practices, such as tillage and diversified crop rotations, impact microbial activity, organic matter turnover, and ultimately soil C and N sequestration. The objectives of this study were to determine the impacts of tillage on soil microbial biomass, mineralized C and N, and soil organic C (SOC) and N (SON) contents for different cotton (Gossypium hirsutum L.) cropping systems in a south-central Texas silt loam soil. Reduced tillage (RT) in a continuous cotton monoculture increased SOC by 24% and SON by 27% compared to conventional tillage (CT) at 0-5 cm, but tillage had no effect at 5-15 cm. Crop rotation increased soil C and N contents compared to continuous cotton, as a cotton-corn (Zea mays L.) rotation under CT increased SOC by 28% and SON by 26% at 0-5 cm compared to CT continuous cotton. Soil organic C and SON were both 18% greater for cotton-corn than continuous cotton at 5-15 cm. For the 0-15 cm depth interval, the CT cotton-corn rotation increased SOC by an average of 518 kg C ha-1 yr-1 and SON by 57 kg N ha-1 yr-1 compared to CT continuous cotton. Cotton under RT sequestered 254 kg C ha-1 yr-1 and 33 kg N ha-1 yr-1 more than cotton under CT. Reduced tillage increased soil microbial biomass C (MBC) by an average of 11 and 18% compared to CT continuous cotton and the cotton-corn rotation, respectively, while microbial biomass N (MBN) for RT was 62% greater than for CT. Tillage decreased mineralized C and N at both depth intervals, while cotton-corn showed higher mineralized C than continuous cotton. Soils for cropping systems that sequestered the most C and N also had the highest microbial biomass and mineralized C and N, indicating close relationships between microbial activity and soil C and N sequestration.