Consensus does not exist for which cost forms (i.e., one accounting solely for explicit cost and the other for both explicit and opportunity costs as in relative opportunity cost) are used in calculating return on investment (ROI) for conservation-related decisions. This research examines how the cost of conservation investment with and without inclusion of the opportunity cost of the protected area results in different solutions in a multi-objective optimization framework at the county level in the Central and Southern Appalachian Region of the USA. We maximize rates of ROI of both forest-dependent biodiversity and economic impact generated by forest-based payments for ecosystem services. We find that the conservation budget is optimally distributed more narrowly among counties that are more likely to be rural when the investment cost measure is relative opportunity cost than when it is explicit cost. We also find that the sacrifice in forest-dependent biodiversity per unit increase in economic impact is higher when investment cost is measured by relative opportunity cost rather than when measured by explicit cost. By understanding the consequences of using one cost measure over the other, a conservation agency can decide on which cost measure is more appropriate for informing the agency’s decision-making process.