Sunday's joint prayer took place at the Church of the Holy Sepulchre, revered in Christian tradition as being built on the site of the crucifixion, burial and resurrection of Jesus.
While praying together for further unity between the eastern and western branches of Christianity, the two leaders knelt side by side on the Stone of the Anointing, where the body of Jesus is said to have been laid before burial.
Earlier, Francis and Bartholomew I, the spiritual leader of the world's Orthodox Christians, signed a landmark pledge to pursue a common dialogue reaffirming values common to the Catholic and Orthodox churches.
"Our fraternal encounter today is a new and necessary step on the journey towards the unity... of communion in legitimate diversity," the declaration reads.
The pledge was signed in the presence of representatives of 13 Catholic and Orthodox churches in Jerusalem.
The meeting was billed as the main reason for the pope's three-day trip to the Middle East. It commemorated the historic rapprochement between both branches of the Christian church 50 years ago, when Pope Paul VI met and embraced Patriarch Athenagoras.
That encounter marked the first easing of tension between the Churches since the Great Schism in the 11th century, which split Rome from Constantinople, the seat of Orthodoxy.
Earlier on Sunday, the pope made an unscheduled stop at the separation barrier Israel is building across the West Bank, and held a silent prayer.
The wall is a major source of contention between Israel and the Palestinians, with Israel saying it is necessary for security purposes, while Palestinians see it as a bid to grab land.
The 77-year-old pontiff also used a speech earlier in the day to express "profound sadness" at Saturday's deadly attack at the Jewish Museum in Brussels and call for an end to anti-Semitism.
tj/dr (AFP, dpa)