Resumo. Abstract Aim: This study records Gyrodactylus neotropicalis and Anacanthocotyle anacanthocotyle in new hosts: Astyanax lacustris and Moenkhausia forestii, on the upper Paraná river floodplain. Besides, ecological aspects of the parasites distribution are explored. Methods Both host species were collected between March and September 2014; monogeneans were removed from the gills using a stereo-microscope and mounted in Hoyer or stained with Gomori’s trichrome and mounted in Balsam of Canada. Illustrations were prepared on a phase contrast microscope. Monogeneans were identified using the appropriate literature. Prevalance, abundance, mean intensity and intensity range were calculated to parasite species in each host. Results Gyrodactylus neotropicalis and Anacanthocotyle anacanthocotyle are recorded in new geographic area, upper Paraná River floodplain, in new host species, A. lacustris and M. forestii and in new infection site, gills. A. anacanthocotyle occur in higher prevalence, abundance and intensity parasitizing A. lacustris, while G. neotropicalis presented higher prevalence, abundance and intensity in M. forestii. Discussion Gyrodactylus neotropicalis described by Kritsky & Fritts, 1970, parasite of Astyanax fasciatus (Cuvier), presented a different shield position between anchors than the specimens studied here; A. anacanthocotyle also described by Kritsky & Fritts, 1970 presented spicules in the cephalic region, what was not observed in the specimens studied here. Plasticity in parasitism may explain the difference between monogenean in different hosts. Conclusion Until now, there was no record of this Gyrodactylidae species parasitizing Characidae fish in South America. Those parasites are the first Gyrodactylidae monogeneans described from A. lacustris and M. forestii.