It was the conventual church of the Hospitallers, immediately adjacent to the Palace of the Grand Master, and presided over by the order's most senior religious official.

At least six Hospitaller grand masters were buried in the church, including Fabrizio del Carretto, whose elaborate funerary slab was placed in its central part.

The church became famous for its collection of relics, which included objects associated with John the Baptist, Jesus, the Virgin Mary, and various other saints.

The building was damaged by two earthquakes in the nineteenth century, and destroyed on 6 November 1856 by a lightning strike which ignited gunpowder stored in its cellars, killing at least 200 people.

Construction seems to have finished under Foulques's successor, Hélion de Villeneuve, who was Grand Master until 1346 and whose coat of arms was carved into the church's north wall.

[7] Underground spaces were constructed beneath the main structure, one of which led to the loggia (covered gallery) between the church and the grand master's palace; these may variously have been crypts, storage rooms or chapels.

[8] The historian Sofia Zoitou has characterised the architecture and contents of the Church of St John as "eloquently articulat[ing] the Knights' political and religious essence".

[18] Between 1435 and 1439, the Spanish traveller Pero Tafur visited Rhodes,[19] and noted that the church was filled with relics, and used both for religious services and for meetings of the Hospitallers.

[20] Between the fourteenth and the sixteenth century, the church was mentioned in several guides for western European pilgrims, who would use Rhodes as a stop on journeys to the Holy Land.

[30] Ludwig Ross, a German archaeologist and former ephor-general of antiquities of Greece, visited in 1843, and noted an ancient Greek inscription,[c] several defaced tombs of Hospitaller knights, and images of the Apostles of Jesus in several niches.

[30] The British archaeologist Charles Newton visited the site during his consular service on Rhodes in April 1853, and wrote a description of the building: he commented that the wooden roof was painted in blue and decorated with stars, while stained-glass windows with escutcheons of the knights, noticed by Rottiers in 1828, were no longer present.

[35] The first attested mention of this collection was made by Ludolf von Sudheim, a German priest who visited Rhodes on a journey to the Holy Land between 1336 and 1341.

[35] From the late fourteenth century, prompted by a loss of revenues caused by the Papal Schism of 1378, the Hospitallers took greater care to expand, curate and promote their collection of relics as a means of generating income from pilgrims visiting Rhodes.

The collection became famous: it was described in several pilgrims' accounts, and listed in the Santa parola ('Holy Words'), a litany dating from 1389–1475, read by sailors and outlining the holy sites along the route of their voyage.

[40] The Hospitallers investigated and satisfied themselves of the authenticity of the hand send by Bayezid, and it was housed in a gold reliquary, decorated with pearls and precious stones, donated by d'Aubusson, the grand master.

[53][d] The amateur archaeologist Alfred Biliotti, who was serving as a consular official for the United Kingdom on Rhodes, conducted an impromptu excavation to rescue survivors.

Further archaeological work took place in 1988, after heavy rain caused the collapse of a revetting wall, uncovering a tomb underneath the central part of the church.