A male of Pteronura brasiliensis (Carnivora: Mustelidae) was found dead on the banks of the Rio Negro, in the Pantanal wetlands of Mato Grosso do Sul state, Aquidauana municipality. Two ticks found attached to its skin were morphologically identified as a second-instar nymph of Ornithodoros rostratus (Argasidae) and a male of Amblyomma sculptum (Ixodidae). In order to complement the morphological identification, these tick specimens were subjected to DNA extraction, and tested using PCR assays to confirm the molecular identity the specimens. Also, the tick DNA samples were tested and were negative in the PCR assays for all the pathogens tested. We also examined 30 batches, consisting of 174 individuals of O. rostratus deposited in the Acari Collection of the Butantan Institute, and we found material from four Brazilian states, including one batch containing 2 males and 2 females from Aquidauana, of Mato Grosso do Sul state, collected from the soil. This was therefore the first record of O. rostratus parasitizing P. brasiliensis and the first locality record (Aquidauana). Likewise, A. sculptum is commonly found in the Pantanal and is reported here for the second time parasitizing the giant otter, which is a host little studied regarding the ectoparasites.