Soil microbial activity mediates processes that affect ecosystem functions. Disturbances by management practices may change microbial biomass and activity. The specific objectives were to reveal heterogeneity of chemical, biochemical, and microbiological properties in different management systems and to determine microbial responses to grazing and cultivation in prairie soils. Treatments included (1) heavily grazed, (2) moderately grazed, (3) undisturbed, (4) abandoned from cultivation, and (5) cultivated with continuous winter wheat or cotton. The undisturbed site has been preserved for at least 50 years, while the rest have been conducted for at least 30 years. The experimental sites are in rolling upland mixed prairie in the Great Plains of United States. Samples were taken along a 200 cm transect at 20 cm intervals in each treatment. Microbial biomass C (Cmic) and N (Nmic), and dehydrogenase activity (DH) were determined to indicate soil microbial population and metabolic activity, and soil organic C (total, Corg and dissolved, DOC) to indicate capacity of a soil to support microbial life. Of soil properties tested, Cmic and Nmic were the most spatially variable, especially in cultivated soils, while Corg varied less than Cmic. DOC varied most in grazed systems. Along transects, greater heterogeneity was shown in cultivated soils when compared with grazed or undisturbed soils. Overall, management practices were responsible for 33 to 98% of total variation in tested soil properties. Microbial activities were greater in undisturbed, abandoned, or grazed systems when compared with those in cultivated ones, indicating partial restoration of the soil ecosystem. However, negative impact of cultivation on soil microbial community in abandoned soils was detectable, suggesting that 30 years of preservation was not long enough to erase human impact on a soil ecosystem. Of those tested, grazed systems are more sustainable based on responses from soil microbial community.