Mr Macron was visiting the Church of St Anne in Jerusalem's walled Old City on Wednesday, where the French flag has flown ever since it was gifted to France’s Napoleon III by the Ottomans in 1856.
It is seen as a provocation in France for Israeli police to enter the church’s sandstone complex, which is considered a French territory.
In a video of the incident, Mr Macron can be seen jostling in the centre of a crowded group in an archway leading to the building and angrily demanding Israeli security personnel to leave the church.
“Go outside. I'm sorry, you know the rules. Nobody has to provoke, nobody,” the president told security personnel.
“We had a wonderful walk, you did a great job in the city and I do appreciate it, but please respect the rules as they are for centuries. They will not change with me, I can tell you,” he added.
The incident has drawn comparisons to a similar confrontation between Jacques Chirac, then-French president, and Israeli security agents at the same church in 1996.
Mr Chirac lost his patience with Israeli officers during a walk in the Old City and described his treatment as a “provocation” before threatening to cut short his trip to the country.
“What, do you want me to go back to my plane and go back to France? Is that what you want?” he asked a security chief.
The then-president refused to enter the church until Israeli police left the site after he complained security was heavy-handed.
An Israeli police spokesperson initially declined to comment on the incident on Wednesday, and an Israeli government spokesperson did not immediately provide comment on behalf of the Shin Bet internal security agency, which also helps guard foreign dignitaries. They later said in a joint statement that it had been agreed in advance that a police officer and Shin Bet guard would escort Macron inside the church.
Mr Macron was visiting Israel to attend Thursday’s World Holocaust Forum at the Yad Vashem Holocaust memorial centre in Jerusalem to commemorate the 75th anniversary of the liberation of Nazi Germany’s Auschwitz death camp.
French diplomats had cautioned that they wanted to avoid mishaps like his predecessors on the president’s first visit to the country.
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“In the current context, France is determined that Iran never acquires a nuclear weapon, but also that we avoid all military escalations in the region,” he said after his discussion with Mr Netanyahu.
The French president’s visit to St Anne was seen as a symbolic stop underscoring Paris’ historical influence in the region.
Additional reporting by Reuters