Fleas are ectoparasitic insects of many birds and mammals. More than 200 species and subspecies of fleas have been recorded from South America; however, they have been poorly studied in Uruguay. In the present study, Polygenis (Neopolygenis) atopus, Polygenis (Neopolygenis) massoiai, Polygenis (Polygenis) axius axius, Craneopsylla minerva minerva, and Sternopsylla distincta are reported for the first time in Uruguay. These fleas parasitize wild rodents, marsupials, and bats, and most of the ectoparasite–host associations are recorded for the first time. Results obtained are consistent with findings in nearby Argentinean and Brazilian localities.