Declines and extinctions are increasing globally and challenging conservationists to keep pace with biodiversity monitoring. Organisms leave DNA traces in the environment and this free DNA in soil, water, and air is referred to as environmental DNA (eDNA). The analysis of eDNA is a highly sensitive method with the potential to rapidly assess local diversity and the status of threatened species. We searched for DNA traces of 30 target amphibian species of conservation concern, at different levels of threat, using an environmental DNA metabarcoding approach to analyze water samples from six montane sites in the Atlantic Coastal Forest and adjacent Cerrado grasslands of Brazil. We successfully detected DNA traces of four declined species (Hylodes ornatus, H. regius, Crossodactylus timbuhy, and Vitreorana eurygnatha); two locally disappeared (Phasmahyla exilis and P. guttata); and one species that has not been seen since 1968 (Megaelosia bocainensis). We confirm the presence of species undetected by traditional methods, underscoring the efficacy of eDNA metabarcoding for biodiversity monitoring at low population densities, especially in megadiverse tropical sites, and its potential application in conservation biology.