The pontiff's efforts to improve relations with the Georgian Orthodox Church suffered a setback after leaders of the denomination chose not to send an official delegation to the Mass and encouraged Orthodox believers to stay away from the service.
The Pope's convoy was met by a small group of hardline Orthodox protestors after his arrival to the city on Friday.
"It's typical proselytising," said Father David Klividze, who was among about 100 people protesting outside the sports stadium.
"Can you imagine how it would be if a Sunni preacher came to Shiite Iran and conducted prayers in a stadium or somewhere else? Such a thing could not be. Therefore, we are speaking against this."
Prior to the Pope's visit to Tbilisi, the Vatican had said that the Mass - held at the Meskhi sports stadium - would be attended by the Orthodox Patriarchate as a "sign of the rapport between the two churches".
On Saturday, the patriarchate updated a statement on its website saying: "as long as there are dogmatic differences between our churches, Orthodox believers will not participate in [Catholic] prayers."
Christianity in Georgia
Christians make up two thirds of population
3 million Orthodox Christians
40,000 Catholics
The Meskhi sports stadium, capacity 27,000, was expected to be filled for the Mass but according to reports only a few thousand people attended the service.
Addressing the crowd, Pope Francis urged pilgrims to find consolation in God and not be "saddened by the lack of harmony around us".
"It is when we are united, in communion, that God's consolation works in us," he said.
The Pope's official visit to Georgia lasted three days. On Sunday, he briefly stopped in Muslim-majority Azerbaijan where it was reported he received a warm welcome from President Ilham Aliyev.
Georgia is a majority Orthodox country. Less than 3% of the population are Catholic according to Vatican statistics.
Relations between Georgia and the Vatican have been tense since the visit of Pope John Paul II to Georgia in 1999 where he attempted to form a treaty granting the Catholic Church legal status in the Orthodox country.