They addressed a protest in Parliament Square, central London, attended by more than 1,000 people.
Conservative contender for City Hall, Zac Goldsmith, warned it would be "catastrophic". Labour's Sadiq Khan called it "madness".
A Heathrow spokesman said a third runway was needed to keep London "at the heart of the global economy".
Mr Goldsmith told the crowd: "We know that our air pollution problems in London would be unsolvable if we expand Heathrow. And we know it requires the demolition of more than 1,000 homes. It is a catastrophic price to pay."
Mr Khan told BBC London: "It would be madness to build a new runway. People who care about London and the health of London, who worry about the noise, who worry about the infrastructure, are united against it."
Green candidate Sian Berry, Liberal Democrat Caroline Pidgeon and UKIP's Peter Whittle all gave speeches against the proposed expansion, joined by environmental campaigners and local residents.
John McDonnell, the shadow chancellor and Labour MP for Hayes and Harlington, also addressed the crowd. He said: "In my constituency at the moment, people are literally dying. They're dying because the air has already been poisoned by the aviation industry."
A Heathrow spokesman responded: "London's future success as a global city, and as a centre for finance and investment, will depend on the strength of links with existing and new markets.
"The Airports Commission has confirmed that Heathrow expansion will keep the capital and the whole of the UK at the heart of the global economy, meet environmental targets and balance the needs of local communities."
Heathrow was chosen by the Davies Commission as the best way to increase airport capacity for London and the South East because it was predicted it would add Â£147 billion in economic growth and 70,000 jobs by 2050.
Prime Minister David Cameron is due to announce a decision on expansion by the end of the year.
Rival mayoral candidates have united to condemn plans for a third runway at Heathrow Airport.