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Background: Vaccination and importation of dogs and cats are prohibited in the Galapagos, resulting in a uniquely isolated population. The purpose of this study was to determine the prevalence of infectious diseases of dogs and cats that impact their health, could spill over to native wildlife, or sentinel diseases of concern to humans., Hypothesis: The isolation of dogs and cats in the Galapagos protects them from diseases common in mainland populations., Animals: Ninety-five dogs and 52 cats presented during a neutering campaign., Methods: A prospective cross-sectional study was performed. Blood was collected for serological and DNA evaluation of a panel of infectious diseases., Results: Antibodies against parvovirus (100%), parainfluenza virus (100%), adenovirus 1/2 (66-67%), and distemper virus (22%) were present in dogs. Dirofilaria immitis was also common in dogs (34%), with lower prevalences of Wolbachia pipiens (22%), Bartonella sp. (13%), Ehrlichia/Anaplasma spp. (1%), and Mycoplasma haemocanis (1%) observed. Antibodies against panleukopenia virus (67%), Toxoplasma gondii (63%), calicivirus (44%), and herpesvirus 1 (10%) were detected in cats. Feline leukemia virus antigen, feline immunodeficiency virus antibody, or coronavirus antibodies were not detected. Bartonella sp. (44%) infections were common in cats, but only one was infected with M. haemofelis., Conclusions and Clinical Importance: Despite their relative seclusion from the rest of the world, cats and dogs of Isabela were exposed to many pathogens found in mainland South America. Parasite prophylaxis, neutering, and strict enforcement of animal movement restrictions would control a majority of the diseases. In the absence of vaccination, a reservoir of susceptible animals remains vulnerable to new disease introductions.