"Lord, enlighten the leaders of the nations, so they may have the courage to abandon projects of death," the pontiff said in the video message in Thursday.
"Today we lift up our prayer for peace in the world, asking that nations renounce weapons and choose the path of dialogue and diplomacy," he said.
Leo releases a video message each month to announce his prayer intentions for that month. The pope's intention for March is "for disarmament and peace".
It was unclear if Thursday's video was created specifically to respond to the US-Israeli military campaign, which has set off a regional war with Iranian attacks in Israel, the Gulf and Iraq, and Israeli attacks in Lebanon.
The Vatican did not immediately respond to a question about when the video was recorded.
The Vatican's top diplomat warned on Wednesday that the US-Israeli strikes undermined international law and said nations did not have a right to launch "preventive wars", an unusually direct criticism of the military campaign.
"If states were to be recognised as having a right to 'preventive war' ... the entire world could risk going up in flames," Cardinal Pietro Parolin, the Vatican's secretary of state, said in an interview with Vatican News.
In his video, the pope asked God to help the world understand "that true security does not come from control fuelled by fear but from trust, justice and solidarity among peoples".
