Tories and Liberal Democrats said parts of the Bill would give sweeping powers to John Prescott, Deputy Prime Minister and Secretary of State for the Environment.
More than 250 clauses in the proposed legislation were designed to prevent an independent-minded mayor such as Ken Livingstone from carrying out radical policies, they said.
Simon Hughes, the Liberal Democrat MP, who used the Commons debate to declare that he would stand for mayor, said the Bill would give Mr Prescott "Henry VIII"-style control over the new Greater London Authority (GLA).
Fears that Mr Livingstone, MP for Brent East, might seek to turn the mayoralty into a new power base had led to the creation of the clauses designed severely to restrict its remit, said Mr Hughes.
One key section would allow the Environment Secretary to take from the mayor the proceeds of proposed "congestion charges" on London's roads.
Although the Government said that it would protect such proceeds for the first 10 years of the GLA, Mr Hughes pointed out that the Bill gave it the power to rescind the promise.
Other "Livingstone clauses" meant that the mayor would have to devise strategies in line with national policies and that the Environment Secretary could severely restrict mayoral spending.
Opening debate on the second reading of the Bill, Mr Prescott said the GLA would bring back the first city-wide administration since abolition of the Greater London Council in 1986 - providing badly needed accountability and leadership
He said: "It will strip away the shadowy committees, the burgeoning bureaucracies and quangos created by our predecessors and give Londoners back their voice."
The new authority would have "tremendous power and responsibility", and its relationship with the Environment Secretary would resemble the present relationship between central and local government.
The GLA would control a budget worth several billion pounds, have strategic powers over transport and economic development and would oversee a new, democratically accountable police authority.
He added: "The next stage of devolution, if you like, might and would hopefully be towards regional government."