Abstract The Corubal (Guinea-Bissau) is a wild but underexplored river in West Africa. This study underscores the potential of environmental DNA (eDNA) surveys to fill biodiversity knowledge gaps in the region. We filtered large water volumes at 11 sites along the watershed, amplified multiple molecular markers, and performed high PCR (polymerase chain reaction) replication and in-depth sequencing. We recorded 2589 amplicon sequence variants, with accumulation curves indicating the need for additional sampling to achieve a thorough survey. The taxonomic assignments were constrained by the scarcity of genomic resources. We recorded 125 species of aquatic and terrestrial vertebrates, including 21 new to the country, predominantly fish (61.9%). Surprisingly, crocodiles were not detected, despite their known presence. There were 11 imperiled species, two of which are Critically Endangered (Western chimpanzee and the mussel Pleiodon ovatus). Our findings support the conservation importance of the Corubal, provide a baseline for future monitoring, and highlight the challenges and opportunities of eDNA surveys in remote tropical rivers.