Abstract The Miocene aquatic and terrestrial fossil record from western Amazonia constitute a clear evidence of the palaeoenvironmental diversity that prevailed in the area, prior to the establishment of the Amazon River drainage. During the Miocene, the region was characterized by a freshwater megawetland basin, influenced by episodic shallow-marine incursions. A fossil vertebrate collection from the middle Miocene strata of the Pebas Formation is here studied and described. This historical collection was recovered in 1912 along the banks of the Itaya River (Iquitos, Peru), during a scientific expedition led by two scientists of the University of Zurich, Hans Bluntschli and Bernhard Peyer. Our findings include a total of 34 taxa, including stingrays, bony fishes, turtles, snakes, crocodylians, and lizards. Fishes are the most abundant group in the assemblage (~ 23 taxa), including the first fossil record of the freshwater serrasalmids Serrasalmus , and Mylossoma , and the hemiodontid Hemiodus for the Pebas system, with the latter representing the first fossil be discovered for the entire Hemiodontidae. The presence of a representative of Colubroidea in the middle Miocene of Iquitos supports the hypothesis of arrival and dispersal of these snakes into South America earlier than previously expected. This fossil assemblage sheds light on the palaeoenvironments, and the geographical/temporal range of several aquatic/terrestrial lineages inhabiting the Amazonian region.