Corn stover as well as perennial grasses like switchgrass (Panicum virgatum) and miscanthus are being considered as candidates for the second generation biofuel feedstocks. However, the challenges to biofuel development are its effects on the environment, especially water quality. This study evaluates the long-term impacts of biofuel production alternatives (e.g., elevated corn stover removal rates and the potential land cover change) on an ecosystem with a focus on biomass production, soil erosion, water quantity and quality, and soil nitrate nitrogen concentration at the watershed scale. The Soil and Water Assessment Tool (SWAT) was modified for setting land cover change scenarios and applied to the Iowa River Basin (a tributary of the Upper Mississippi River Basin). Results show that biomass production can be sustained with an increased stover removal rate as long as the crop demand for nutrients is met with appropriate fertilization. Although a drastic increase (4.7–70.6%) in sediment yield due to erosion and a slight decrease (1.2–3.2%) in water yield were estimated with the stover removal rate ranging between 40% and 100%, the nitrate nitrogen load declined about 6–10.1%. In comparison to growing corn, growing either switchgrass or miscanthus can reduce sediment erosion greatly. However, land cover changes from native grass to switchgrass or miscanthus would lead to a decrease in water yield and an increase in nitrate nitrogen load. In contrast to growing switchgrass, growing miscanthus is more productive in generating biomass, but its higher water demand may reduce water availability in the study area.