In human-transformed landscapes, predators may feed on domesticated animals, and thus affect human well-being, creating negative perceptions and leading to conflict with people that can result in the persecution of the predator. We studied the factors that influence the perception of the Black-and-chestnut Eagle (Spizaetus isidori) in four rural Andean communities in Colombia and compiled historical and recent evidence on the persecution and other causes of mortality of this species in the country. We applied 267 questionnaires and conducted 16 interviews with local inhabitants, via visits to homes and schools in the surroundings of S. isidori nests. The perception of S. isidori by the inhabitants was largely negative and influenced by different socio-demographic factors such as gender, chicken (Gallus gallus) ownership, and chicken management. The records we obtained indicate that 47 eagles were shot, 16 were captured (three for illegal trafficking) and two were electrocuted on high-tension wires. The persecution of S. isidori occurs as retaliation or as a preventative measure against chicken predation, and is a significant cause of mortality of this species in Colombia. Effective conservation planning for S. isidori in Colombia needs to go further than the protected areas system, and include a socioecological perspective in conservation practices applied at landscapes scales that are dominated by people. Education programs and socioecological research, along with participatory work in local communities are key to the conservation of S. isidori in breeding territories. This approach can also prevent conflict over food resources—G. gallus and other poultry—that are shared by humans and S. isidori in rural landscapes.