The ABC reported the comedian's Tuesday night show at the ICC Sydney Theatre had to be paused after the demonstrators, who had a Palestinian flag, interrupted the Jewish actor.
Two members of the audience shouted at Seinfeld before security intervened and audience members booed.
But Seinfeld responded to them and acknowledged their "strong political feelings".
"But you don't know where to say them," he said.
"You think that ruining the night, it doesn't affect me, all these people, you're ruining their night."
The protesters were escorted out of the venue.
According to the ABC, Seinfeld then made light of the incident.
"I love them, too, they just need a little direction of, you know, where to use their tremendous brain power."
It was the second on-stage interruption during Seinfeld's live world tour, which includes a show at Christchurch's Wolfbrook Arena on Wednesday, June 26.
He was also interrupted on Sunday night by audience members playing social media videos of pro-Palestinian chants, such as "From the River to the Sea, Palestine will be free", the ABC reported.
The audience members were also escorted out of the venue.
Seinfeld has previously talked about how excited he is to perform in Auckland and Christchurch next week.
“I am very excited to return to New Zealand for my first ever performance in Christchurch and to see more of such a beautiful country," he said.
Seinfeld has spoken about his support for Israel since the October 7 Hamas attack in which more than 1100 people were killed and dozens more taken captive, according to Israeli officials.
Two months after the attack, Seinfeld went on a solidarity trip to Israel, the ABC reported.
Since October 7, more than 37,300 Palestinians have been killed and about 85,500 injured, according to the Gaza Health Ministry.
