Conservation return on investment (ROI), for all spatial scales, varies according to a wide range of characteristics. One feature that makes conservation ROI at the parcel level different from larger scale ROI is the impact of parcel size variation on ecological and economic effectiveness. Protected area size maintains an important role in both the benefit and cost associated with conservation. However, few studies have explicitly focused on the role of protected area size on conservation ROI specifically at the parcel level. Therefore, conservation ROI has been limited in its effectiveness in prioritizing parcels for conservation. The objective of our research is to examine how protected area size influences a parcel’s ecological and economic effectiveness through conservation ROI. This objective is accomplished by analyzing the parcel- level acquisition cost and the conservation benefit of protected areas acquired by a conservation organization, The Nature Conservancy (TNC). We develop an empirical model to examine how differences in protected area size influence conservation benefit as an ecological measurement, and how differences in the conservation benefit, as a measurement of conservation value, subsequently alter conservation cost. By assessing the sequential relationship in a spatial econometric modeling framework, we first examine the consequence of the size variation on conservation ROI and then we calculate and rank the ROI for each protected area with and without considering the spatial spillovers of conservation benefit and acquisition cost. We found that (1) protected areas acquired by TNC create more connected habitat, thereby improving species protection and mobility in the existing protected area network that existed prior to the TNC acquisition, and subsequently, such improvement is a major impetus to determine acquisition cost, (2) the increase in effective mesh size per dollar invested to acquire a parcel is greater for larger parcels than smaller parcels, implying that the overall efficiency that considers both ecological and economic efficiencies is higher for protecting larger areas relative to smaller ones, and (3) the inclusion of spillovers of both conservation benefit and cost in the ROI decision-making tool provides information about which parcels’ locations affect conservation benefit and cost in their neighboring parcels and to what extent.