SignificanceKnowledge of resource and habitat use by wildlife is essential to support conservation actions. Stable isotope analysis (SIA) is a useful method for the acquisition of this type of information. Samples for SIA can be obtained through indirect and noninvasive methods, which is favorable for studies of threatened species. We used SIA to compare the resource and habitat use and trophic structure of mammals between preserved areas and human-modified landscapes in a tropical rainforest. Our study shows that mammals in human-modified landscapes present an altered trophic structure and use food items from the agricultural matrix, while they depend on forest resources in preserved areas. Our findings stress the need for favorable management of the agricultural matrix to support wildlife survival.