Founded by Cardinal Federico Borromeo, the library was among the first in Lombardy to be accassible to the public. Borromeo wanted it to be an educational centre. He commissioned books and manuscripts from Western Europe, Greece and Syria. He even made a series of acquisitions of whole libraries – he acquired all manuscripts from the Benedictine monastery of Bobbio and the collection of the Paduan Vincenzo Pinelli – over 800 manuscripts including the illuminated Iliad called 'Ilia Picta'.

The library focuses on history, religion and literature of the classical period. Its rich collection includes the Arab and Oriental works of extreme cultural importance. The glottological-dialectological collection of Carlo Salvioni and the landmark library of Eugenio Casanovav will be a real treat to all bookworms. Varied palimpsests sit next to various meticulously illustrated manuscripts such as the 'Libro d'ore Borromeo' and the 'Gellio'. The Ambrosiana also features many valuable bindings of both manuscripts and prints, one of which is in human skin.

During his stays in Rome, Cardinal Borromeo thought of new improvements to his collection.He wanted it to be accessible for all scholars and to serve as a bastion of Christian scientific thought balancing the Protestant works. To provide space for the 15,000 manuscripts and around 30,000 printed books, he ordered for a new library to be build in 1603. When Sala Fredericiana (the reading room), was opened in 1609, it was second only to the Bodlein Library in Oxford. Cardinal Borromeo also envisioned an academy and a collection of pictures, which required a new building – the future Pinacoteca.

Soon after the death of its founder, the library acquired twelve manuscripts of Leonardo da Vinci, among them the 'Codex Atlanticus'. Today, the collection comprises around 12,000 drawings by European artists, from the 14th through to the 19th Century. They have been donated by numerous patrons, artists, collectors, academicians, art dealers, and architects.