In March 1530, Malta and Gozo were handed over to the Sovereign Military Order of the Knights of Saint John of Jerusalem by the mightiest monarch of the age, the Holy Roman Emperor Charles V.
Initially, the rule of the Knights brought no improvements to Gozo. In July 1551, Iċ-Ċittadella was besieged by the Turks, led by Sinam Pasha and, the most feared and famous of all the corsairs, Rais Dragut. The medieval walls without flanks and terreplein to resist gunpowder bombardment were easy prey to the besiegers and, on 26 July, the weak fortifications succumbed. The Matrice was ransacked. Around 500 managed to escape by letting themselves down on ropes; the rest, about 5000, were taken into slavery.
Grandmaster Juan de Homedes and his Council initially entertained the idea of abandoning Iċ-Ċittadella. Yet sweet home soon attracted back those that had escaped and the few who amassed enough money to be ransomed from slavery. By September 1554, the Matrice was functioning again. The stouthearted Gozitans soon restored their church and their houses and persuaded the Council to change its mind.
Iċ-Ċittadella and its Matrice flourished once again. On 6 June 1623, Bishop Baldassarre Cagliares established a Collegiate Chapter within the church. Pope Alexander vii reaffirmed this first Collegiate of Gozo on 20 October 1663.
A collegiate church is one served by a body of secular clergy who are bound to the daily chanting or recitation of the Divine Office in choir.
By this time the structure of the church had deteriorated but it was not easy to build a new one. After the siege of 1551, the economy of Gozo was shattered and the population decimated: both the financial means and the people who could make offerings and help manually in such a major project were wanting. Around 1680, the population reached the pre-1551 level again and plans for a new church began to be entertained.