Soils managed under native ecosystems relative to agricultural row crops often have significant differences in soil physical and microbial properties. Assessment of relationships among these properties may provide useful information in how the physical environment affects microbial properties. The objective of this research was to quantify soil physical properties and soil enzyme activity, physiological and molecular characteristics for native, restored, and cultivated prairies. Soil physical properties included bulk density, pore-size distributions, saturated hydraulic conductivity and water-stable aggregates. Sampling sites were located on Mexico silt loam (fine, smectitic, mesic Aeric Vertic Epiaqualfs). Land treatments included native, uncultivated prairie with established warm-season grasses and forbs; 10-yr-old restored prairie dominated by sericea lespedeza ( Lespedeza cuneata); 10-yr-old restored prairie dominated by little bluestem (Schizachyrium scoparium ), side-oats gramma (Bouteloua curtipendula), and Indian grass (Sorghastrum nutans); a 14-yr-old conservation reserve program site with cool-season grasses and low density forage legumes; and a site under long-term row crop production with the past 14 years under a corn (Zea mays)-soybean (Glycine max) rotation cropping system. Research demonstrated that soil measurements based on soil enzyme activity, physiological and molecular characteristics, and selected physical traits (water-stable aggregation, saturated hydraulic conductivity) differentiated soils managed as native prairie, restored prairie, or cultivated land. Results indicated that sites under restoration to prairie vegetation are transitional between native prairie and cultivated soils based on combined physical and microbiological analyses. The relationship of soil organic matter (SOM) to biological activity is illustrated by strong correlations between soil DNA (representing the bacterial community) and water stable aggregates (r² = 0.82) and glucosidase activity (representing SOM decomposition; r² = 0.83). The use of physiological and molecular analyses of prairie soils yielded new insights on the complex functional and structural diversity of their soil bacterial communities, which contribute to the biological characteristics of these soils.