With peace agreed to and with Barberini power-brokers dead or exiled, the citizens of Castro were left alone. But Odoardo Farnese, who had signed the original peace accord, died in 1646 and was succeeded by his son Ranuccio II Farnese, Duke of Parma.
In 1649, Ranuccio refused to pay Roman creditors as his father had agreed in the treaty signed five years prior. He also refused to recognize the new bishop of Castro, Monsignor Cristoforo Giarda, appointed by Pope Innocent X. When the bishop was killed en route to Castro, near Monterosi, Pope Innocent X accused Duke Ranuccio, and his supporters, of murdering him.
In retaliation for this alleged crime, forces loyal to the Pope marched on Castro. Ranuccio attempted to ride out against the Pope's forces but was routed by Luigi Mattei. On 2 September, on the Pope's orders, the city was completely destroyed. Not only did Pope Innocent's troops destroy the fortifications and general buildings of Castro, they destroyed the churches as well so as to completely sever all links between the city and the papacy. As a final insult, the troops destroyed Duke Ranuccio's family Palazzo Farnese and erected a column reading Quì fu Castro ("Here stood Castro").
Duke Ranuccio II was forced to cede control of the territories around Castro to the pope, who then attempted to use the land to settle debts with the Ranuccio's creditors. This marked the end of the Second War of Castro and the end of Castro itself – the city was never rebuilt.