The application of environmental DNA (eDNA) metabarcoding as a biomonitoring tool has greatly increased, but studies have focused on temperate aquatic macro‐organisms. We apply eDNA metabarcoding to detecting the mammalian community in two high‐biodiversity regions of Brazil: the Amazon and Atlantic Forests. We identified Critically Endangered and Endangered mammalian species and found overlap with species identified via camera trapping. We highlight the potential for using eDNA monitoring for mammals in biodiverse regions and identify challenges: we need a better understanding of the ecology of eDNA within variable environments and more appropriate reference sequences for species identification in these anthropogenically impacted biomes. Mammalian families (shown with stylised drawings of representative species within hexagons) identified from environmental DNA samples in the Amazon and Atlantic Forests of Brazil. Water and sediment samples were taken from rivers (‘Meeting of the Waters’; top left) and streams (bottom left and right) and subjected to DNA metabarcoding analyses using mammal‐specific primers. A total of 28 molecular operational taxonomic units (MOTUs) were identified, and 13 of these could be identified to species level. This case study has highlighted the potential of this non‐invasive genetic technique to detect (and potentially monitor) terrestrial and aquatic mammals in these biodiverse Neotropical regions.