The species of the genus Phoneutria (Ctenidae), also called banana spiders, are considered amongst the most venomous spiders in the world. In this study we revalidate P. depilata (Strand, 1909), which had been synonymized with P. boliviensisis (F.O. Pickard-Cambridge, 1897), using morphological and nucleotide sequence data (COI and ITS-2) together with species delimitation methods. We synonymized Ctenus peregrinoides, Strand, 1910 and Phoneutria colombiana Schmidt, 1956 with P. depilata. Furthermore, we designated Ctenus signativenter Strand, 1910 as a nomen dubium because the exact identity of this species cannot be ascertained with immature specimens, but we note that the type locality suggests that the C. signativenter syntypes belong to P. depilata. We also provide species distribution models for both species of Phoneutria and test hypotheses of niche conservatism under an allopatric speciation model. Our phylogenetic analyses support the monophyly of the genus Phoneutria and recover P. boliviensis and P. depilata as sister species, although with low nodal support. In addition, the tree-based species delimitation methods also supported the separate identities of these two species. Phoneutria boliviensis and P. depilata present allopatric distributions separated by the Andean mountain system. Species distribution models indicate lowland tropical rain forest ecosystems as the most suitable habitat for these two Phoneutria species. In addition, we demonstrate the value of citizen science platforms like iNaturalist in improving species distribution knowledge based on occurrence records. Phoneutria depilata and P. boliviensis present niche conservatism following the expected neutral model of allopatric speciation. The compiled occurrence records and distribution maps for these two species, together with the morphological diagnosis of both species, will help to identify risk areas of accidental bites and assist health professionals to determine the identity of the species involved in bites, especially for P. depilata.