A video circulating on social media showed ultra-Orthodox Jews spitting on the ground as pilgrims carried crosses along Jerusalem's Via Dolorosa – the route Christians believe Jesus walked before being crucified.
On Tuesday, Mr Netanyahu, whose coalition government, including ultra-Orthodox and far-right parties, is one of the most right-wing in Israel's history, said: “I strongly condemn any attempt to inflict harm on worshippers, and we will take urgent steps against such actions.
“Offensive behaviour towards worshippers is a desecration and is unacceptable. We will show zero tolerance towards any harm to worshippers.”
Mr Netanyahu did not reference a specific attack in the message.
The video followed the recent publication of similar footage of people insulting or acting aggressively towards Christians in the Old City.
After capturing it in 1967, Israel annexed East Jerusalem, including the Old City, in a move never recognised by the international community.
The Old City remains at the heart of the Palestinian-Israeli conflict as well as tensions between the world's three major monotheistic faiths.
Last month the Latin patriarch of Jerusalem, Archbishop Pierbattista Pizzaballa, said that while attacks on Christians in the Old City were “not a new phenomenon”, they had been more frequent “in the recent period”.
Mr Pizzaballa, who Pope Francis anointed as a cardinal on Saturday, said there were many reasons for the increase, including education.
“There are some movements, some rabbis also, who are inciting on this, or at least approving of this,” he said.
“We have not to forget the past relations between Jews and Christians were not simple, to be diplomatic, and all this creates this context.”
The archbishop also said that the frequency of “this phenomenon … is connected, temporarily at least, with this [Israeli] government”.
On Tuesday, the rabbi of the Western Wall, Shmuel Rabinowitz, condemned in “the strongest terms the violence against believers in the Old City and all forms of violence”.
“We must do everything in our power to preserve the delicate fabric of the Old City,” he said, addressing “the leaders of all religions”.