Treefrogs of the genus Osteocephalus have been the focus of several taxonomic and phylogenetic studies, especially in the last two decades. These recent studies have shown that the diversity of this charismatic Amazonian genus is still largely underestimated. Through the evidence of morphological and molecular data, we describe a new species of the Osteocephalus alboguttatus species group from the Purus-Madeira Interfluve, southwestern Brazilian Amazonia. The new species differs from other Osteocephalus by having a small body size (snout–vent length 32.1–44.1 mm), skin texture non-sexually dimorphic, dorsum smooth with a few scattered small tubercles, vocal sac single and subgular, frontoparietal ridges not externally visible, and a dark tan brown iris with lighter vermiculation. The rapid increase in the number of new frog species described from the Purus-Madeira Interfluve highlight the importance of sampling poorly explored and remote areas in Amazonia, as well as the value of supporting taxonomic research to accelerate species documentation in face of the biodiversity crisis.