The Mayor was being questioned on transport, crime and other issues facing Londoners when the crowd began demanding he resign.
In an attempt to stop the chanting, the chair of the event Tony Devenish was forced to threaten to "clear the room", saying: "This is your final warning".
One audience member heckled the mayor, shouting: "You should be in a nappy."
WATCH: People's Question Time audience chant 'get Khan out'
The comment came as Khan attempted to address the cost of living crisis - in which Khan mentioned people being unable to afford nappies.
He was also defending his free school meals policy, which saw the Mayor allocate £135million of emergency funding to the project.
Khan said: "We've got to have policies that support families that are really struggling, and we know half the families in London are just about getting by, or are really struggling.
"And that's why free school meals are really helping."
The event saw Khan defend controversial policies such as the Ultra Low Emission Zone (Ulez) expansion across London.
He told the event: "More than half of Londoners with asthma live in outer London.
"Twenty-four of the 30 GPS with the largest number of asthma patients are all in outer London.
"Nobody put up with dirty water, we shouldn't put up with dirty air."
One audience member dismissed Ulez as "appalling", claiming it was a ploy to "replenish TfL's budget".
Others also criticised Sadiq Khan with one accusing him of telling "lies" about the Ulez expansion.
In a furious rant directed at the London mayor, one audience member claimed Ulez is "closing" London.
But Khan said he has put in "big changes" to support Londoners with non-compliant cars, including putting "more money" towards the scrappage scheme, which provides financial assistance to help eligible London residents scrap vehicles that don't meet the Ulez emissions standards.
One audience member said: "I have a friend who is going to see his disabled sister every weekend out of London in his non-compliant car. It's now costing him 1300 pounds per year to go and see and help her.
"It's costing other people lots and lots of money as well. You say London is open, but your policies are closing it more and more every day.
"I believe Ulez is actually replenishing TfL's budget. I don't believe the lies."
She added: "So is it really about making money for TfL and Mr Khan - all the millions you're making from poor people like me, I have an old car.
"What are you going to spend this money on?"
Khan responded: "So in relation to the support for those who've got non-compliant cars, we've made progress since the last people's questions which is now every single family in London is eligible for support through the scrappage scheme.
"Every single business is now eligible for support. Small businesses, every single charity, is now eligible for support. Those who are disabled are eligible for a grace period.
"Those are the big changes we've made. We've put in more money for the scrappage scheme."