Tropical agricultural frontiers are rapidly changing and present unique opportunities to reconcile ecosystem services (ES) provision, biodiversity conservation, and livelihood maintenance of rural communities. To understand the dynamics of these frontiers, we analyzed preferences, reasons associated with importance, and spatial perceptions of ES and biodiversity in Mexico at two scales. In seven workshops and 42 interviews, participants ranked preferred ES and expressed reasons for their importance. They identified the relevant areas for ES and biodiversity. We used this data to create an index to obtain maps of perceived spatial patterns and perform a cluster analysis to identify ES bundles. A redundancy analysis highlighted the factors that help explain the spatial patterns and qualitative and quantitative methods were used to explore the reasons behind participants’ rankings of relative importance. We found two contrasting bundles at the regional scale: the natural protected area, where biodiversity is highest, and the agricultural frontier, where agricultural services were highest. At the ejido scale, ejidos whose livelihoods are mainly centered on agriculture and those that develop diverse activities were grouped. Socio-ecological factors (e.g., ejido area, PES program, and indigenous population) further distinguished ejidos focused on agricultural expansion, those dominated by marginalized local inhabitants, and those focused on forest management. The most dominant reasons underlying ES rankings include having sufficient nutritious food and economic support linked to agricultural services. We discuss the role of conservation strategies, historical government decisions, and biophysical characteristics in shaping strong trade-offs between biodiversity conservation and agriculture—and the role of voluntary local conservation initiatives and a national PES scheme in supporting opportunities to reconcile some of these trade-offs. This study demonstrates the importance of integrating local community preferences and perceptions into spatially explicit products that can be used in inclusive conservation and landscape planning approaches for tropical landscapes worldwide.