Soil management practices influence soil physical and chemical characteristics and bring about changes in the soil microbial community structure and function. In this study, the effects of long-term conventional and no-tillage practices on microbial community structure, enzyme activities, and selected physicochemical properties were determined in a continuous corn system on a Decatur silt loam soil. The long-term no-tillage treatment resulted in higher soil carbon and nitrogen contents, viable microbial biomass, and phosphatase activities at the 0–5 cm depth than the conventional tillage treatment. Soil microbial community structure assessed using phospholipid fatty acid (PLFA) analysis and automated ribosomal intergenic spacer analysis (ARISA) varied by tillage practice and soil depth. The abundance of PLFAs indicative of fungi, bacteria, arbuscular mycorrhizal fungi, and actinobacteria was consistently higher in the no-till surface soil. Results of principal components analysis based on soil physicochemical and enzyme variables were in agreement with those based on PLFA and ARISA profiles. Soil organic carbon was positively correlated with most of the PLFA biomarkers. These results indicate that tillage practice and soil depth were two important factors affecting soil microbial community structure and activity, and conservation tillage practices improve both physicochemical and microbiological properties of soil.