Évora was definitively reconquered from Arab hands in 1166 by Geraldo Sem Pavor (Gerald the Fearless), and soon afterwards the new Christian rulers of the city began to build a cathedral, dedicated to the Virgin Mary. This first building, built between 1184 and 1204, was very modest and was enlarged circa 1280-1340, this time in early Gothic style. The cathedral received several valuable additions through time, such as the Gothic cloisters (14th century), the Manueline chapel of the Esporão (early 16th century) and a new, magnificent main chapel in baroque style (first half of the 18th century). It is the largest of the mediaeval cathedrals in Portugal, and one of its best examples of Gothic architecture.

It is common belief that flags of the fleet of Vasco da Gama on his first expedition to the Orient, were blessed in the first presbytery of the cathedral in 1497.

The most important historical figure associated with the cathedral was Cardinal-King Henrique (1512–1580), who was archbishop and cardinal of Évora. Cardinal Henrique, who was brother of King John III, had to succeed D. Sebastião as King of Portugal after his death in the Battle of Alcácer-Quibir. The Cardinal-King ruled only between 1578 and 1580.

In the 16th and 17th centuries, the Cathedral of Évora was the setting of the so-called School of Évora of polyphony, which played an important role on the music history of Portugal. Composers related to the Cathedral include Mateus de Aranda and Manuel Mendesand his pupils, Duarte Lobo and Filipe de Magalhães.