Cash can no longer be used on any of London's buses in a move that Transport for London (TfL) says will save £24m a year.
Passengers will need a prepaid or concessionary ticket, Oyster card or a contactless payment card to travel.
TfL said only 0.7% of all bus journeys were paid for with cash and that tourists were unlikely to be affected.
But the Green Party said more ticket outlets were needed and questioned how vulnerable passengers would cope.
TfL said its research showed the move, which came into force at 04:30 BST, was unlikely to affect tourists "as the vast majority use a prepaid ticket, such as a Visitor Oyster, to get around the capital".
Its drivers have been asked to guide vulnerable people.
Last month TfL introduced the "one more journey" feature for Oyster users, allowing passengers to make one further trip if they have insufficient credit on their card, which TfL said had benefitted around 44,000 customers a day.
Mike Weston, TfL's director of buses, said: "Removing cash from our bus network not only offers customers a quicker and more efficient bus service but it enables us to make savings of £24m a year which will be re-invested to further improve London's transport network."
But Green Party London Assembly member Darren Johnson said questions remained over whether passengers who lost or damaged their Oyster cards would be allowed to travel at all, saying that more than 2,100 Oyster cards were lost, stolen or stopped working on the average day last year.
Mr Johnson said: "I am very concerned that TfL are only planning 19 new Oyster ticket stops in preparation for the cashless switchover. This seems highly inadequate, especially when every ticket office across the tube network is going to be closed.
"I also have serious concerns about the impact which this sweeping change could have on vulnerable passengers."