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Environmental DNA (eDNA) metabarcoding has emerged as one of the most efficient methods to assess aquatic species presence. While the method can in theory be used to investigate nonaquatic fauna, its development for inventorying semi-aquatic and terrestrial fauna is still at an early stage. Here we investigated the potential of aquatic eDNA metabarcoding for inventorying mammals in Neotropical environments, be they aquatic, semi-aquatic or terrestrial. We collected aquatic eDNA in 96 sites distributed along three Guianese watersheds and compared our inventories to expected species distributions and field observations derived from line transects located throughout French Guiana. Species occurrences and emblematic mammalian fauna richness patterns were consistent with the expected distribution of fauna and our results revealed that aquatic eDNA metabarcoding brings additional data to line transect samples for diurnal nonaquatic (terrestrial and arboreal) species. Aquatic eDNA also provided data on species not detectable in line transect surveys such as semi-aquatic, aquatic and nocturnal terrestrial and arboreal species. Although the application of eDNA to inventory mammals still needs some developments to optimize sampling efficiency, it can now be used as a complement to traditional surveys., (© 2021 John Wiley & Sons Ltd.)