The Opera di Santa Maria del Fiore was born in 1296 with the foundation of the new Cathedral, and its administration passed to the Wool Merchants Guild in 1331. Since its foundation, the Opera has been an independent lay institution with the task of attending to the Cathedral patrimony. Since then, it has preserved the complex and, by an agreement with the Chapter of the Florentine Metropolitan, attends to the expenses of worship. In 1777, a grand-ducal decree entrusted the Opera also with the Baptistery and various other buildings. With the Concordat of 1929, the Institution assumed the status of "Fabbriceria" (works commission).

The Museum was instituted in 1891 to preserve the artefacts not housed inside the Cathedral, along with various objects remaining from the work site. In 2001, the Museum was restructured and the collections received new layouts. The scientific artefacts Opera is entrusted with also include the medieval solstice marble of the Baptistery, the mechanical clock, the gnomons of the Cathedral, and various materials used for experiments on the rotation of the earth in the 19th century.

The collection comprises various pieces of work site equipment (hoists, lifts, hempen ropes, etc.) and scientific instruments, which can be dated as of the Middle Ages, and are exhibited in the Museum’s "Brunelleschi’s Work Site" room, along with wooden models of the Cathedral.

On the top floor of the Museum is the study room for the visually impaired, containing reproductions of works and models that can be handled by visitors.