Evaluating the occurrence of diseases in aquatic mustelids is a priority in the conservation strategies for the Neotropical river otter and giant otter. Thus, the objective of this study was to determine the frequency of infection caused by Cryptosporidium spp. and Giardia sp. in both host species in northern Brazil. The collection of biological samples was carried out in the states of Amapá, Amazonas, Pará and Rondônia, totalling 337 faecal samples of these species, which were processed using Kinyoun's technique for the identification of Cryptosporidium spp. oocysts, and centrifugal flotation in zinc sulphate solution for visualization of Giardia sp. cysts. All samples were also tested by direct immunofluorescence. The frequency of infection by Cryptosporidium spp. was higher than Giardia sp., in the two otter species. In the analysed samples co-infection by both protozoa was also found in 4.47% (14/313) of Neotropical river otter and 20.83% (5/24) of giant otter samples. Oocysts and cysts of Cryptosporidium and Giardia, respectively, may remain infectious within specific environmental conditions for long periods of time. The current identification of Neotropical and giant otters as hosts of these protozoa increases the possibility of infection in this species and the transmission of those agents to other aquatic and terrestrial organisms, as well as to human populations. The findings of this study represent the first description of Cryptosporidium spp. and Giardia sp. affecting Lontra longicaudis and Pteronura brasiliensis.