The rainforests of the Neotropics shelter a vast diversity of plant, animal and microscopic species that provide critical ecosystem goods and services for both local and worldwide populations. These environments face a major crisis due to increased deforestation, pollution, and climate change, emphasizing the need for more effective conservation efforts. The adequate monitoring of these ecosystems has proven a complex and time consuming endeavour, which depends on ever dwindling taxonomic expertise. To date, many species remain undiscovered, let alone described, with otherwise limited information regarding known species population distributions and densities. Overcoming these knowledge shortfalls and practical limitations is becoming increasingly possible through techniques based on environmental DNA (eDNA), i.e., DNA that can be obtained from environmental samples (e.g. tissues, soil, sediment, water, etc.). When coupled with high-throughput sequencing, these techniques now enable realistic, cost-effective, and standardisable biodiversity assessments. This opens up enormous opportunities for advancing our understanding of complex and species-rich tropical communities, but also in facilitating large-scale biomonitoring programs in the neotropics. In this review, we provide a brief introduction to eDNA methods, and an overview of their current and potential uses in both terrestrial and aquatic ecosystems of neotropical rainforests. We also discuss the limits and challenges of these methods for our understanding and monitoring of biodiversity, as well as future research and applied perspectives of these techniques in neotropical rainforests, and beyond.