Infections with Entamoeba spp. are recognized as a cause of clinical disease in many species including humans and reptiles; however, cases in amphibians are under-reported. Investigation of a mortality event among a captive population of Cranwell’s horned frogs Ceratophrys cranwelli at a production facility in Florida, USA, revealed that deaths were due to the newly described Entamoeba species CT1. Infection caused severe necroulcerative gastroenterocolitis with a predilection for the colon. To date, this Entamoeba species has only been described in invasive cane toads Rhinella marina in Australia. Retrospective screening of archived anuran cases from a zoological pathology service identified 8 cases from captive populations that had histological evidence of gastrointestinal entamoebiasis. Molecular characterization was positive in 3 cases. Two cases, 1 in a Puerto Rican crested toad Peltophryne lemur and 1 in an Amazon milk frog Trachycephalus resinifictrix, showed 100% homology to E. ranarum and 1 case in a White’s tree frog Litoria caerulea showed 100% homology to Entamoeba sp. CT1. This is the first report of novel Entamoeba sp. CT1 being associated with clinical disease in anurans within North America and also the first report of this Entamoeba species causing disease within managed collections as far back as 2003.