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High‐diversity mixtures of perennial tallgrass prairie vegetation could be useful biomass feedstocks for marginal farmland in the Midwestern United States. These agroenergy crops can help meet cellulosic agrofuel targets while also enhancing other ecosystem services on the landscape. One proposed advantage of high‐diversity prairie biomass feedstocks is that they should become nutrient limited at a slower rate than monoculture feedstocks. In this study, we examine rates of soil nutrient depletion and the physiology and performance of a focal species (switchgrass, Panicum virgatum L.) in four prairie agroenergy feedstocks with different species composition and diversity. The feedstocks in this study were a 1‐species switchgrass monoculture, a 5‐species mixture of C4 grasses, a 16‐species mixture of C3 and C4 grasses, forbs, and legumes, and a 32‐species mixture of C3 and C4 grasses, forbs, legumes, and sedges. To assess feedstock effects on soil, we measured changes in soil N/P/K over a five‐year period. We also performed a greenhouse study, in which we grew switchgrass plants in field soil conditioned by each feedstock. To assess feedstock effects on plant function, we measured four physiological traits (photosynthetic rate, chlorophyll concentration, leaf florescence, leaf N concentration) on switchgrass plants within each feedstock in the field. In the soil analysis, we found that the 5‐species feedstock displayed higher rates of soil N/P/K depletion than other feedstocks. In the greenhouse analysis, we found that switchgrass plants grown in soil conditioned by the 5‐species feedstock were smaller than plants grown in soil conditioned by other feedstocks. In the physiological analysis, we found that switchgrass plants in the 5‐species feedstock had lower leaf N, photosynthesis, chlorophyll concentration, and higher florescence than switchgrass plants growing in other feedstocks. Collectively, our results show that prairie agroenergy feedstocks with different species composition and diversity have different rates of soil nutrient depletion, which influences the physiology and performance of plants within the feedstock. These differences would ultimately impact the ecosystem services (e.g., biomass production, need for fertilizer) that these prairie agroenergy feedstocks provide. [ABSTRACT FROM AUTHOR]