Two ectoparasites, spiny rat mite, Echinolaelaps echidninus (Berlese) (n = 27), and spined rat louse, Polyplax spinulosa (Burmeister) (n = 2), were recorded for the first time in Yucatán State and documented with photographs. The ectoparasites were identified mostly on black rat, Rattus rattus (L.), with serological evidence of flavivirus infection. Adult female E. echidninus was most common and found on black rat with serological evidence of yellow fever, Modoc, and Apoi viruses. We also reported for the first time the cat flea, Ctenocephalides felis (Bouché), on R. rattus. We discussed the potential for the arthropods to bite and transmit pathogens to humans.