A new species of Bryophryne from the humid montane forest of the Department of Cusco, Peru, is described. Specimens were collected at 2795–2850 m a.s.l. in the Área de Conservación Privada Ukumari Llaqta, Quispillomayo valley, in the province of Paucartambo. The new species is readily distinguished from all other species of Bryophryne by having green coloration on dorsum, and blue flecks on flanks and ventral parts. Specimens are characterized by lacking a distinct tympanic annulus, tympanic membrane, and dentigerous processes of vomers, and by having dorsal skin shagreen, discontinuous dorsolateral folds, skin tuberculate on flanks, skin areolate on ventral surfaces of the body, and fingers and toes without lateral fringes or webbing. The new species has a snout–vent length of 14.2–16.9 mm in three males and 22.2–22.6 mm in two females, and is smaller than all other congeneric species except for B. abramalagae. Generic allocation is supported by low genetic distances of the 16S mitochondrial gene and morphological similarity with other species of Bryophryne, and geographic distribution. Bryophryne phuyuhampatu sp. n. is only known from the type locality, a cloud forest along the Quispillomayo River in the upper Nusiniscato watershed.