The landmark 2019 Intergovernmental Science-Policy Platform on Biodiversity and Ecosystem Services (IPBES) Global Assessment cited land-use change as the primary driver of biodiversity loss. The 2016 peace agreement in Colombia has led to increasing agricultural expansion into biodiversity-rich forests. We have focused on the case of Colombia to demonstrate an approach to maximize the biodiversity benefits from limited conservation funding while ensuring that landowners maintain economic returns equivalent to agriculture. We applied a quantitative model that relates conservation investment to national biodiversity outcomes. Then we identified six regions with high potential return on investment by spatially modelling the risk of forest conversion and the expected impact of conservation actions. Our results suggest that agricultural expansion, left unchecked, would increase national biodiversity loss by 38–52% by 2033, and that doubling investment is necessary to counteract this loss. Our approach can be broadly used to target investment to weigh development and biodiversity goals. We demonstrate the approach in Colombia with its accelerated social and environmental changes and show how the efficiency of conservation options can be improved by considering opportunity cost of conservation to communities whose livelihoods depend on agriculture. This approach can be applied to other contexts to examine development and policy priorities to estimate financial needs for achieving biodiversity goals.