The Colosseum underwent significant transformations in its use during the medieval era. In the late 6th century, a modest church was erected inside the amphitheater structure, although this addition does not seem to have conferred a religious meaning to the entire building. The arena, meanwhile, was converted into a cemetery, and the wide spaces between the arches and under the seats found new purposes as workshops and shelters. According to historical records, these spaces were even leased until the 12th century.

During the pontificate of Gregory the Great, numerous ancient monuments passed under the guardianship of the Church, which then held the predominant authority. However, due to a lack of resources, the Church was unable to maintain them adequately, and as a result, these monuments fell into disuse and were subject to looting. In the Middle Ages, the decline of the city affected all the vestiges of the Roman Empire. Earthquakes in 801 and 847 inflicted severe damage to a virtually abandoned building on the edge of the medieval city.

In 1084, when Pope Gregory VII was banished from Rome, several monuments passed into the hands of the city's influential noble families, who used them as fortifications. Around 1200, the Frangipani family took possession of the Colosseum and fortified it, treating it as a castle and establishing their dominion over it. The Colosseum changed owners several times until it was returned to the Church in 1312.

The devastating earthquake of 1349 inflicted substantial damage to the Colosseum's structure, resulting in the collapse of the south side of its external facade. Many of the detached stones were recovered and used in the construction of palaces, churches (including buildings in Vatican City), hospitals and other buildings throughout Rome. A religious order settled in the northern portion of the Colosseum and inhabited it until the early 19th century. The stone inside the amphitheater underwent intense quarrying, either for reuse elsewhere or, in the case of the marble facades, for the production of quicklime by burning. The bronze brackets that held the masonry were removed from the walls, leaving behind numerous marks. These scars can still be seen in the structure of the building today.