Reclamation practices following open-pit mining typically include the reconstruction of soil-like profiles using a combination of native soil materials, industrial by-products, and fertilizers. Our overall objective was to compare the quality of eight soil materials used during reclamation in the Athabasca oil sands region of western Canada by characterizing their microbial communities as well as their response to a range of fertilization treatments. Materials included two carbon-rich surface soil materials, four B horizons with varying extractable phosphorus and pH, the parent geological material (PGM), and tailings sands (TSS), a by-product of oil extraction. Measured indices of microbial activity included the activities of b-glucosidase, acid phosphatase, and phenol oxidase. Total biomass and structure of the soil microbial community were characterized based on phospholipid fatty acid (PLFA) analysis. Soil materials and fertilization treatments were tested with multivariate regression trees and non-metric multidimensional scaling. Material type, rather than fertilization level, had the largest impact on all microbial parameters, including biomass, activity, and composition. Only the nutrient-poor materials (PGM, TSS, and one of the B horizons) showed a response to fertilization. The microbial composition of three of the four B horizons was more similar to the two carbon-rich surface soil materials than it was to PGM or TSS. Hence, we propose that these subsoil materials present an advantage over the use of the underlying PGM when reconstructing upland sandy soils. Finally, results indicated that soil microbial biomass could be used as a good indicator of seedling growth when no fertilizer was applied.