Geological explorations of the basal beds of the Río Yuca Formation (Tucupido region, Portuguesa State, western Venezuela) resulted in the recognition of a new vertebrate assemblage that includes eight taxa: the toxodont cf. Adinotherium, a Peltephilidae armadillo, the freshwaters fishes Platysilurus and Phractocephalus, the caiman Purussaurus, an indeterminate dolphin, turtles, and the previously recognized sloth Pseudoprepotherium venezuelanum. When compared with the higher latitudes faunas of Argentina and Chile, the presence of cf. Adinotherium and peltephilids in the Rio Yuca Formation is consistent, but not conclusive, with a Santacrucian to Frisian SALMA age. The associated fauna, as well recent apatite fission track analysis, indicates that the Río Yuca assemblage is more likely younger in age, specifically Middle to Late Miocene. So far, the Miocene localities of the northern part of South America have provided a less prolific fossil record compared to the southern part of the continent (e.g., Santacrucian and Friasian faunas of Patagonia), but the present work documents the surprising occurrence of two taxa (Peltephilidae and Nesodontinae) common in southern high latitude faunas of South America, implying the persistence of the Santacrucian-Friasian genus Adinotherium in younger strata from northern South America, and that peltephilids were much more widespread during the Miocene than previously recognized. The presence of these common Patagonian taxa (Peltephilidae and Nesodontinae) in Río Yuca also supports the hypothesis of prior researchers for the existence of biogeographical connections between the northern and southern portions of South America during the Late Oligocene or Early Miocene, which facilitated faunal interchange between the two regions. Finally, the biogeographical affinities of the freshwater fishes and the giant caiman (Purussaurus) indicate close relationships of the Tucupido region with the ancestral distribution of the Orinocoan–Amazonian drainage system.