On 8 September 1514, the army of the Grand Duchy of Lithuania, led by Grand Hetman Konstanty Ostrogski, defeated a much larger Muscovite force at the Battle of Orsha. Ostrogski was an Orthodox magnate fighting for a Catholic king, Sigismund I, and the victory carried weight on both sides of the religious divide. He kept a vow he had made before the battle, funding the construction of an Orthodox monastery in Vilnius dedicated to the Holy Trinity. For nearly a century the monastery anchored Orthodox life in the Lithuanian capital. Then came the Union of Brest in 1596, and within years the church and monastery were transferred to the Uniates, who answered to Rome while keeping Eastern liturgy. The monks of the Basilian Order made it their main house in the Grand Duchy.

Three side chapels were added in the early seventeenth century, each funded by a noble family who wanted to be buried beneath it. In one chapel rests Barbara Naruszewicz, who died in 1627, beneath a marble gravestone carved by Italian masters. Another chapel was funded in 1622 by Eustachy Korsak-Hołubicki, a vocal supporter of the Union, whose family produced Rafajil Korsak, later Metropolitan of Kyiv. Inside the church survives a Renaissance tombstone for the Vilna burgomaster Othanasius Braha and his son Antony, dated 1576, with Cyrillic inscriptions and floral ornament. From 1715 to 1915 the church housed the Vilna icon of the Theotokos Hodegetria, said by tradition to have been painted by Saint Luke and brought to Vilnius when Helena of Moscow married Grand Duke Alexander Jagiellon. In 1915, with German armies advancing, the icon was evacuated. Where it ended up, no one knows.

In 1821, the tsarist authorities expelled the Basilian monks and converted part of the monastery into a prison. Two years later, in October 1823, a young Vilnius University student named Adam Mickiewicz was arrested along with other members of the Filaret Association, a secret patriotic society of students who studied Polish literature and dreamed of restoring the partitioned Commonwealth. They were held inside the former monastery for months, and the experience marked Mickiewicz for life. He drew on his imprisonment in Forefathers' Eve, which became one of the foundational works of Polish Romanticism. The cell where he was held is now a small chapel of remembrance. Mickiewicz left Vilnius in 1824 under sentence of internal exile and never returned.

After surviving Soviet suppression and decades of changing jurisdictions, the Holy Trinity church returned to the Order of Saint Basil the Great and the Ukrainian Greek Catholic Church. Today, services are conducted entirely in Ukrainian, an unusual rite to find in central Vilnius and a marker of how thoroughly the Ukrainian diaspora and the historic Greek Catholic tradition have outlasted the empires that once tried to absorb or extinguish them. The painted depiction of Saint Josaphat Kuntsevych, the Greek Catholic archbishop killed by an Orthodox mob in 1623 and later canonized, still presides above the altar; near it, a plaque commemorates the three Lithuanian Martyrs whose original Orthodox community lay just up the street. The same building has held both sides of that history. It still does.