During observations at a breeding colony of Humboldt Penguins (Spheniscus humboldti), we noted five male penguins enter nests occupied by breeding pairs and engage in aggressive interactions with the resident adult. These nest intrusions resulted in egg loss or infanticide, and accounted for 11.1% of breeding failure in the colony. Two intruder males ultimately mated with respective resident females. This behavior had not been observed previously in Humboldt Penguins and we suggest that it may occur when males are unable to obtain mates through advertising at the nest. Intrusion behavior may slow down population recovery in this endangered species.