Francesco Niccolini, son of the once ambassador to Rome Giovanni Niccolini, was groomed for a church career, but at the death of his father in 1611 abandoned the priesthood and some years later, in 1618, married Caterina di Francesco Ricciardi. Niccolini was appointed ambassador to Rome on April 15, 1621 and remained there until 1643. On returning home he was nominated Gentleman of Court by Ferdinando II (1610-1670) and in 1647 became Chancellor of the Order of Santo Stefano.

Niccolini met Galileo while the latter was on his mission to Rome in 1624, and the two formed a close friendship. Niccolini greatly helped Galileo, especially in the period prior to the publication of his Dialogo sopra i due massimi sistemi del mondo [Dialogue Concerning the Two Chief World Systems] (Florence, 1632). On invitation of the Grand Duke's Secretary Andrea Cioli (1573-1641), he was introduced to the Master of the Holy Palace, Niccolò Riccardi (1585-1639), in charge of revising the work, so as to speed up the process of publication.

In the months following the publication of the Dialogue, Francesco Niccolini was constantly in contact with both Andrea Cioli and Galileo himself, keeping them abreast of the shifting humors at Rome. During his stay in Rome during the trial, Niccolini assisted Galileo with great affection, to the point of offering to cover his living expenses personally should the Grand Duke ever reneg on his commitment. In the period following Galileo's condemnation, Niccolini did all he could to get him pardoned. Though failing in that aim, he did succeed in getting permission for him to reside in Florence.