Although once considered a ‘counterfeit paradise’, the Amazon Basin is now a region of increasing interest in discussions of pre-colonial tropical land-use and social complexity. Archaeobotany, archaeozoology, remote sensing and palaeoecology have revealed that, by the Late Holocene, populations in different parts of the Amazon Basin were using various domesticated plants, modifying soils, building earthworks, and even forming ‘Garden Cities’ along the Amazon River and its tributaries. However, there remains a relatively limited understanding as to how diets, environmental management, and social structures varied across this vast area. Here, we apply stable isotope analysis to human remains (n = 4 for collagen, n = 17 for tooth enamel), and associated fauna (n = 61 for collagen, n = 28 for tooth enamel), to directly determine the diets of populations living in the Volta Grande do Rio Xingu, an important region of pre-Columbian cultural interactions, between 390 cal. years BC and 1,675 cal. years AD. Our results highlight an ongoing dietary focus on C₃ plants and wild terrestrial fauna and aquatic resources across sites and time periods, with varying integration of C₄ plants (i.e. maize). We argue that, when compared to other datasets now available from elsewhere in the Amazon Basin, our study highlights the development of regional adaptations to local watercourses and forest types.