Toxoplasma gondii is one of the most common protozoan parasites of humans and warm-blooded animals. Members of the family Felidae are the only definitive hosts of this parasite and, thus, important in the epidemiology of the disease. Previous studies on Pacific islands have found T. gondii infections in a number of avian species where domestic cats (Felis catus) have been introduced. Little is known about T. gondii in the Galapagos Islands, although introduced domestic cats in the archipelago are known to be T. gondii antibody-positive. In this study, we quantified prevalence of antibody to T. gondii in two threatened avian marine species, Galapagos Penguins (Spheniscus mendiculus) and Flightless Cormorants (Phalacrocorax harrisi), and tested the hypothesis that this parasite is more prevalent on Isabela Island (with cats) than on Fernandina Island (without cats). Overall, antibody prevalence was 2.3% in both Galapagos Penguins and Flightless Cormorants from samples collected during 2003–2005, and in 2008. In Galapagos Penguins (n=298), a significantly higher antibody prevalence was found in penguins on Fernandina Island (free of cats) than on Isabela Island (with cats; Fisher's exact test; P=0.02). In Flightless Cormorants (n=258), there was a higher antibody prevalence in cormorants living on Isabela than on Fernandina, although this difference was not statistically significant (Fisher's; P=0.19). This study is the first to show exposure to T. gondii in endemic avian species in the Galapagos Islands, providing evidence for disease-related risks associated with the feral cat population in the archipelago. We provide possible explanations for these findings and recommendations for future studies towards a better understanding of the epidemiology of T. gondii in the Galapagos Islands.