Commuters faced "annoyance" and disruption on Friday, as climate change campaigners blocked major London roads during rush-hour.
Extinction Rebellion Lewisham "swarmed" roads in the south-east of the capital in an attempt to put pressure on the Government over air quality.
Air pollution in the borough of Lewisham was found to be approximately six times higher than safe levels recommended by the World Health Organisation in 2017.
The recommended amount of PM2.5 particles, which can cause asthma and other respiratory problems, is 25 micrograms per cubic metre of air; however, highs of 150mg were recorded in Lewisham from October 2016 to September 2017.
Holly, 34, who did not give her surname, was caught up in traffic jams after protesters blocked the A205 South Circular in Catford, the A21 at Lewisham station, and the A2 in Deptford, until about 9am.
While stuck in her car outside Lewisham station, "annoyed" Holly said: "It's backed up and I've been sat in it for more than half an hour. "I'm definitely going to be late for work now."
The death of a nine-year-old child who lived in the area is to be re-examined at an inquest, to determine whether it was linked to air pollution. Ella Kissi-Debrah, who lived near the South Circular Road in Lewisham, died in 2013 after having an asthma attack.
A spokesman for Mr Khan said: "The mayor recognises we face a climate emergency and shares the protesters' passion for tackling this issue. But he is clear that causing disruption for Londoners in this way is unacceptable. The protesters' actions caused problems for commuters trying to get to and from work.
"The mayor is doing all he can to reduce the impact of climate change on Londoners - but it is up to the Government to take the bold action required at a national level."
Harry Gibson, 35, took part in Friday morning's action, his second Extinction Rebellion protest.
He said: "The environment catastrophe will far outweigh the damage caused by a few roadblocks on a Friday morning." "We need to look to the future for future generations."