Perennial biomass from grasslands managed for conservation of soil and biodiversity can be harvested for bioenergy. Until now, the quantity and quality of harvestable biomass from conservation grasslands in Minnesota, USA, was not known, and the factors that affect bioenergy potential from these systems have not been identified. We measured biomass yield, theoretical ethanol conversion efficiency, and plant tissue nitrogen (N) as metrics of bioenergy potential from mixed-species conservation grasslands harvested with commercial-scale equipment. With three years of data, we used mixed-effects models to determine factors that influence bioenergy potential. Sixty conservation grassland plots, each about 8 ha in size, were distributed among three locations in Minnesota.