Union delegates have voted in favour of a call for a referendum on the EU treaty, increasing pressure on Prime Minister Gordon Brown to hold one.
Unions say ministers must do more to guarantee workers' rights
Mr Brown says it is not needed because the treaty proposals are very different to the failed EU constitution, on which a referendum had been promised.
But the TUC backed a GMB motion which wants ministers to drop the opt-out on the charter of fundamental rights.
The Tories, UKIP, and some Labour and Lib Dem MPs also back a referendum.
The vote follows another row with Mr Brown over public sector pay, on which the TUC conference has already backed co-ordinated strikes.
'Very bad feeling'
Asked if union delegates had been looking for a fight with the prime minister, TUC General Secretary Brendan Barber said: "No we haven't, but on this issue there are some very strong feelings".
He said the government's insistence on a special protocol on the charter of fundamental rights - which enshrines employment protection including trade union rights - was "totally unjustified" and had caused "very bad feeling".
GMB general secretary Paul Kenny told the BBC: "If we get a referendum and the terms haven't change, it's going to be very difficult to persuade workers to vote for it [the treaty]."
Delegates rejected a separate motion from the RMT union calling for unions to campaign against the treaty in the event of a referendum.
In the meantime, Europe Minister Jim Murphy has been questioned by MPs on the foreign affairs committee, about the "opt outs".
Chairman Mike Gapes, a Labour MP, suggested it would be easier for the government if it did not insist on an opt out of the Charter of Fundamental Rights as one of its "red line" issues - areas on which the government is insisting Britain retains control.
Mr Murphy said while some people were unhappy about it, the government had done "the right thing, in the interests of the UK economy, and in the interests of the deal we struck in June".
He also rejected suggestions from Tory MP David Heathcoat Amory that the "opt outs" were vulnerable to legal challenges from other member states in the European Court of Justice.
Sir Menzies Campbell will not back calls for a referendum
Mr Brown said a poll was not needed because the treaty is not the same as the constitution - on which the Labour government promised a referendum - which was rejected by Dutch and French voters in 2005.
But Conservative leader Mr Cameron has pledged to hold an Opposition Day debate, calling for a referendum, soon after MPs return to Parliament in October.
And Mr Murphy said MPs could have the opportunity to demand a referendum - as had been the case on the Maastricht Treaty, on which there was a vote in Parliament.
Lib Dem decision
He said could not see "any reason why that sort of principle shouldn't be retained and maintained -- but that's an issue for Parliament".
Meanwhile the BBC's Norman Smith has said the Lib Dems will not call for a referendum, after pressure on leader Sir Menzies Campbell from pro-European Lib Dem peers.
The party had called for a referendum on the previous constitution - but has decided the new treaty is substantially different, which will make it harder for pro-referendum campaigners to get a majority in Parliament to put pressure on Mr Brown, he said.