We provide information of the distribution, habitat, and conservation status of the harlequin frog Atelopus seminiferus, a poorly known species from northern Peru. Multiple individuals of A. seminiferus were detected inside the Alto Mayo Protected Forest, San Martin region, 87-98 km northwest from the type locality. Additionally, we used skin swab samples to test for the prevalence of the chytrid fungus Batrachochytrium dendrobatidis (Bd), a pathogen that has been linked with population declines of harlequin frogs throughout tropical America. Our findings represent the first record of A. seminiferus inside a natural protected area, and we recommend an update of the IUCN Red List geographic range map of this species. Though we did not detect individuals infected by Bd, additional surveys are required to further assess the elevational distribution and potential for chytrid fungus infection of this Critically Endangered species.