The soil microbial community plays a key role in many important soil functions, including organic matter and nutrient cycling. These highly complex communities, however, are inherently difficult to study. Soils were collected in 2013 under native prairie and varying cropping systems, including willow, switchgrass, a corn-soybean rotation, corn-soybean-wheat rotation, and a grass and legume mixture in Missouri, USA. Microbial community function was compared by measuring soil dehydrogenase (DHA), β-glucosidase (BGL), β-glucosaminidase (BGA), and fluorescein diacetate (FDA) enzyme activity. Microbial community structure was determined by phospholipid fatty acid (PLFA) analysis. Associated soil quality indicators included soil organic carbon (SOC), active carbon (AC), total nitrogen (TN), and water stable aggregates (WSA). Native prairie exhibited the greatest levels of enzyme activity, SOC, TN, WSA, and microbial biomass as estimated by PLFA analysis. Among cropping systems, willow had significantly greater SOC, TN, BGL, FDA, and DHA, followed by the grass and legume mixture and the remaining cropping systems. In contrast, the grass and legume plots contained the greatest total microbial biomass, followed by willow. In addition, based on PLFA analysis, differences were found among cropping systems in the microbial community structure. Overall, this study demonstrates that cropping systems affect the microbial community structure and function, in addition to traditional indicators of soil quality.