Reclaimed roads on the Little Missouri National Grasslands of southwestern North Dakota have not returned to pre-disturbance conditions. Phospholipid fatty acid analysis was performed on soil samples collected from reclaimed roads and adjacent prairie to assess reclamation effects on the microbial community. Additionally, nutrient cycling capacity was measured by four enzyme assays. Ordination analysis of PLFA data identified a distance gradient indicating microbial communities of reclaimed roads were different from the prairie. Specifically, Gram-negative bacteria and arbuscular mycorrhizal fungi are associated with roads; soil organic matter was associated with prairie sites. Soil enzyme activities associated with prairie sites indicate greater nutrient cycling. The soils of reclaimed roads have not accumulated sufficient organic matter to sustain both plant and microbial communities characteristic of the surrounding prairie.