On 26 January 1625 he was appointed Bishop of Senigallia. On 2 February 1625 he was consecrated bishop by Laudivio Zacchia, Bishop of Corneto e Montefiascone, with Antonio Díaz (bishop), Bishop of Caserta, and Lorenzo Azzolini, Bishop of Ripatransone, serving as co-consecrators. Later he was appointed "Grand Inquisitor" of the Roman Inquisition between 1629 and 1633, Librarian of the Holy Roman Church between 1633 and 1646 and Major Penitentiary between 1633 and 1646. He served as the Camerlengo of the Sacred College of Cardinals twice. He participated in the papal conclave of 1644 which elected his brother's successor, Pope Innocent X. After the Wars of Castro Innocent X launched an investigation into the Barberini family which forced Antonio Barberini's nephews, Francesco Barberini (Senior), Antonio Barberini (Antonio the Younger) and Prince Taddeo Barberini, into exile.