The graffiti, discovered Sunday, was written in Hebrew on an outside wall of the Dormition Abbey and included phrases such as "kill the pagans" and "death to the Christian unbelievers, enemies of Israel".
It was similar to previous acts blamed on Jewish extremists, including arson and vandalism attacks on the same church.
The suspects aged 15 and 16 were to appear in court today, a day after being arrested, police said.
Vatican efforts to negotiate greater rights at the neighbouring Upper Room, where the Last Supper is believed to have occurred, have sparked opposition from nationalist and Orthodox Jews, who revere part of the building as the tomb of King David.
Pope Francis celebrated a mass at the Upper Room during a visit in 2014.
Jewish extremists have targeted Palestinians, Christians and even Israeli military property in "price-tag" attacks - a term that indicates there is a price to be paid for moves against Jewish settlers.