In a video provided by PETA, the two activists wearing black T-shirts proclaiming "bullfighting is a sin" are seen trying to hop over a barricade in St Peter's Square before being stopped by papal security and being led away.
Leo's popemobile, in which the pope had been circling the square ahead of a general audience, briefly paused while the women were intercepted, the video showed.
Leo, the first American pope, heads on Saturday for a seven-day visit to Spain -- which holds about 1,500 bullfights each year.
Opponents call bullfighting barbaric, while fans defend it as a deeply rooted ancient art form.
PETA said in a statement that in Spain "every year thousands of bulls are slaughtered in bullfights held in honour of Catholic saints."
"During these spectacles, lances tear into the bull’s body, banderillas are driven into his back and, weakened and bleeding, he is stabbed through the lungs. Paralysed but often still conscious, he is often mutilated before his body is dragged out of the arena," said PETA.
Bullfighting "stands in stark contrast to Catholic teaching", said the organisation, noting that the late pope Francis wrote in his 2015 "Laudato Si" encyclical that "every act of cruelty towards any creature is contrary to human dignity".
READ ALSO: Inside Spain - Don't expect bullfighting to be banned anytime soon
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