Dozens of passengers tweeted their frustration at their inability to follow government guidance to keep two metres away from others to slow the spread of the illness.
Khan has been at odds with the government over the Tube network, which is operating at around 50% of its normal capacity during peak times.
The London mayor tweeted that "we cannot run more services" as a third of Transport for London (TfL) staff are off sick or self-isolating – but Boris Johnson told MPs on Wednesday it should be possible to "run a better Tube system”.
Latest coronavirus news, updates and advice
Passengers described “packed” services, claiming TfL staff were not managing crowds in line with government guidance on social distancing.
Patricia Pereira posted: "Jubilee line service packed, Waterloo station escalators out of use forcing people to use the ones that are working and be extremely near.
"Three TfL staff by gate line talking, no one manage the crowd (or) helping on social distancing!"
She added: "I am a key worker, I don't want to get ill!"
Others also vented their frustrations at the mayor directly:
However, some passengers stated that their Tube journeys were quieter than normal on Thursday:
Issuing travel advice, Khan tweeted: ”Employers: staff must work from home wherever possible to help protect our key workers who need to travel.
"If you have to go to work, please don't travel at rush hour #COVID19.”
Figures from TfL show that passenger numbers on Wednesday morning were down 92% compared with the same day last year.
Speaking on Thursday morning, Khan said that early-morning Tube use on Thursday was down 13% on the previous day, while bus use was down 8% over the same period.
He tweeted: “Thank you to all Londoners who are following the rules and staying home.
“The more we stay at home the more lives we can save.”