Microbial biomass and enzyme activities are affected by management practices and can be used as sensitive indicators of ecological stability. Microbial biomass C (MBC), microbial biomass N (MBN) and eight enzyme activities involved in the cycling of C, N, P and S were studied in the surface (0–5 cm) of an Irwin silty clay loam soil (fine, mixed, mesic, Pachic Arguistoll) in a tallgrass prairie ecosystem. Treatments of annual spring burning and N fertilization were initiated in 1986 and encompassed: (1) unburned–unfertilized, (2) burned–unfertilized, (3) burned–fertilized, and (4) unburned–fertilized. Activities of dehydrogenase, β-glucosidase, urease, deaminase, denitrifying enzyme, acid phosphatase, alkaline phosphatase, and arylsulfatase were assayed. Long-term burning and N fertilization of the tallgrass prairie soil reduced MBC and MBN relative to the unburned–unfertilized treatment. The effects of burning and N fertilization varied among the enzymes and the time of sampling. Long-term burning significantly (P<0.05) increased activities of urease and acid phosphatase, but decreased activities of β-glucosidase, deaminase and alkaline phosphatase. Long-term N fertilization significantly increased activities of β-glucosidase and acid phosphatase but decreased urease activity. This study found that several soil enzyme activities can be used as indicators of ecological changes caused by N fertilization and long-term burning management practices. The relevance of these changes in surface soil to the long-term sustainability of this ecosystem needs further evaluation.