The Galapagos sea lion (Zalophus wollebaeki), an endangered species, experiences high pup mortality (up to 100%) in years when El Niño events reduce food supply in the Galapagos Islands. Mortality of pups in non-El Niño years is estimated to be 5% in undisturbed colonies. From 2009 to 2012 we observed high pup mortality (up to 67%) in colonies close to the Galapagos capital, Puerto Baquerizo Moreno, where contact with humans, domestic animals, and rats is frequent. Gross postmortem findings from 54 pups included hemorrhagic lesions in liver and congestion in lungs; histopathology suggested a possible association with infectious diseases. Evidence of Leptospira infection was found in five out of seven samples collected in 2010. Canine distemper viral (CDV) RNA was detected in tissues from six sea lions (in 2011–12), four of which were confirmed by nucleotide sequencing. The absence of CDV antibodies in 109 juvenile animals tested in 2014 at urban and remote colonies could indicate that the CDV infection observed in 2011 was likely confined to a few animals. Our results indicated that Galapagos sea lions have been exposed at least to two pathogens, Leptospira and CDV; however, the impact of these infections on the sea lions is unclear.