In the last decades fungal pathogens are causing devastating population declines across a broad range of taxa. A newly emerging fungal disease, sea turtle egg fusariosis, caused by members of the Fusarium solani species complex (FSSC), has been reported to be responsible for hatching failure in sea turtles around the world. However, this has not been reported in other non-marine turtle species. Herein we report high hatching failure from eggs symptomatic of fusariosis in the yellow-spotted Amazon river turtle ( Podocnemis unifilis), inhabiting a pristine environment in the Ecuadorian Amazon. We assessed hatching success from eggs symptomatic and asymptomatic of fusariosis ( n = 680 eggs), tested for Fusarium infection by PCR amplifying the TEF-1α gene (n= 68 turtle internal egg swab samples) and sequenced eight amplicons for screening of FSSC membership on an Illumina Miseq. Hatchability was 72% for asymptomatic eggs, whilst only 8% of symptomatic eggs hatched. Eight percent of asymptomatic and 58% of symptomatic eggs tested positive for Fusarium spp. and sequencing revealed that nine sequence variants from three asymptomatic and four symptomatic eggs corresponded to F. keratoplasticum, F. solani and F. falciforme, the three major FSSC pathogens already reported in sea turtle egg fusariosis. Our study therefore suggests that observed hatching failure of eggs showing symptoms of fusariosis is at least partially caused by Fusarium pathogens within FSSC in a freshwater turtle. This report highlights that fusariosis is more widespread among the Testudines order than previously reported and is not limited to sea environments, which is of particular conservation concern.