Extinction Rebellion (XR) campaigners have demanded the cancellation of the Silvertown Tunnel.
Members of the group walked from The Crystal in Royal Victoria Dock to City Hall in an action timed to coincide with Earth Day (April 22).
XR Newham hoped to deliver a letter to Sadiq Khan, who is standing as the Labour Party candidate for mayor of London, telling him the Silvertown Tunnel must be stopped.
However, their attempt to deliver the message was thwarted at the doors of City Hall, where security guards refused to accept it.
Eden Sophie from the group said: "It would have been great if they had accepted the letter, but we're not giving up on stopping this tunnel."
Fellow activist Mike Bold said: "It's a dreadful legacy to be left by any mayor of London that our children will not be able to breathe."
Members passed the Silvertown Tunnel construction site in North Greenwich before arriving at City Hall. They carried a memorial wreath along the route to highlight the impact of air pollution on public health.
XR argues the scheme will not reduce congestion, will fail to boost local economies and exposes people to more toxic air.
The letter says the tunnel's £2billion cost dwarfs the mayor's flagship £50million green new deal fund, which aims to tackle global warming.
The road tunnel under the Thames would link Silvertown to the Greenwich Peninsula.
Transport for London says the scheme will cut congestion at the Blackwall Tunnel, improve journey times, boost jobs, reduce air pollution on busy roads and increase cross-river bus links
XR's letter adds: "[T]he silent majority, terrified of catastrophic climate change and mass extinctions, are losing faith in Labour’s ability to take decisions to tackle the climate emergency."
London Labour did not respond to a request for comment, but Mr Khan's manifesto includes a pledge for 80 per cent of all journeys in the capital to be made by bike, public transport or on foot.
There are also commitments on clean energy, restoring green spaces and jobs.
XR's action follows campaigners jet-washing "stop the tunnel" onto a River Thames embankment on April 15.
Earth Day is an annual event aimed at highlighting the need for environmental protection.
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