The Magdalena River basin harbors a large biodiversity of vertebrates, with numerous endemic species, many of which are threatened with extinction. Following the same trend as most freshwater ecosystems globally, the Magdalena faces threats from human activities, causing habitat degradation and the decline of its biodiversity. Monitoring the freshwater biodiversity is crucial to detect early impacts and guide conservation actions in this river system, which is of vital ecological importance for Colombia. Here, we used environmental DNA (eDNA) metabarcoding, with two primer sets designed to target mitochondrial DNA regions, specifically the 12S ribosomal RNA gene, targeting fish as well as amphibians, reptiles, birds, and mammals in the Magdalena River. We detected a total of 158 vertebrate taxa, not only aquatic but also terrestrial, arboreal, and aerial. The diversity of these vertebrates increases as the river mouth is approached, accompanied by a change in the composition of the aquatic vertebrate assemblages, with two distinct groups linked to the geomorphology of the lower Magdalena basin. The assemblage was characterized by a high turnover of taxa between the sites closest to the mouth of the river and a marked nesting of taxa when comparing the sites closest to the river mouth with those farther away. We conclude that eDNA metabarcoding allows characterizing vertebrate assemblages in large rivers, assessing conservation status, and elucidating biodiversity patterns with minimal ecosystem disturbance. The information obtained through this approach can contribute to the sustainable management and preservation of vital freshwater ecosystems and their associated biodiversity.