The region was first discovered by Europeans in 1501, and was seen by Christopher Columbus on his fourth voyage in 1503. The first European colony in South America, Santa Maria la Antigua del Darién, was established there by the Spanish in 1510. The settlement was unsuccessful, however, and was soon abandoned. It was from this town that Vasco Nuñez de Balboa made his march to the Pacific in 1513. Some of the refugees from Santa Maria went on to found Panama City in 1519.

There was another attempt to colonize Darién in the seventeenth century, this time by Scottish Protestants. It too ended in failure.