Positive relationships between plant species diversity, soil microbial function and nutrient cycling have been well documented in natural systems, and these relationships have the potential to improve the production and sustainability of agroecosystems. Our objectives were to study the long-term effects of planted species composition and nitrogen (N) fertilization on soil microbial biomass C, extracellular enzyme activity, changes in total soil C, soil fertility and aboveground biomass yield in mixtures of native prairie species managed with and without N fertilizer for bioenergy production at four sites in Minnesota (MN), USA. Species were sown into mixture treatments and composition was not maintained (i.e., no weeding) throughout the duration of the study. Species mixture treatments at establishment included a switchgrass (Panicum virgatum L.) monoculture (SG), a four-species grass mixture (GM), an eight-species legume/grass mixture (LG) and a 24-species high diversity forb/legume/grass mixture (HD). Species diversity and aboveground productivity were similar for most mixture treatments at final sampling after 11 or 12 years of succession. Despite this homogenization of productivity and diversity throughout the study, the effects of planted species diversity and a decade of succession resulted in some differences in soil variables across species mixture treatments. On a peat soil in Roseau, MN, soil enzyme activities including β-glucosidase (BG), cellobiohydrolase (CBH) and phosphatase (PHOS) were highest in HD compared to GM treatments. On a sandy soil at Becker, MN, total soil C increased in all treatment combinations at the 0–15 and 15–30 cm depth intervals, with SG showing greater increases than HD at the 15–30 cm depth. Final soil pH also varied by species mixture at the Becker and Roseau sites, but differences in treatment comparisons varied by location. Nitrogen fertilization did not affect any response variable alone, but interacted with species mixture treatment to influence PHOS and total soil C at Becker. The inconsistent effects of species mixture and N fertilization on soil biological and chemical properties observed across sites highlight the importance of local soil and climate conditions on bioenergy and ecosystem service provisioning of perennial bioenergy cropping systems.