A Tory MP has accused Welsh Labour of wanting to "stop" UK ministers spending money on projects in Wales.
On Wednesday, the Treasury announced it would control a pot of "levelling up" cash for Wales, Scotland and Northern Ireland - as well as in England.
The announcement prompted the Welsh Government to accuse it of "aggressively" undermining devolution.
But Conservative MP Stephen Crabb said he feared Welsh Labour had "thrown their lot in with the nationalists".
The UK Government says its "levelling up" agenda aims to reduce regional inequality in the UK.
The announcement of £4bn for England last year triggered an extra £800m for Wales, Scotland and Northern Ireland.
However, this week the Treasury said it will run the fund for all four UK nations rather than hand any extra cash to the devolved governments.
Now a joint statement by two Welsh Government ministers claims the new policy would mean "little more than £50m each year for Welsh projects" and accuses the Conservative government of having "an appalling record on providing Wales with even a fair share of UK spending".
The statement followed the comments by Mr Crabb, MP for Preseli Pembrokeshire and a former Welsh secretary, made during a St. David's Day debate in Parliament.
He said there was a "clear value" in being part of a UK-led vaccine procurement strategy and said the UK Government had provided "billions of pounds of additional support for Wales".
"That was only possible because at the heart of our United Kingdom there is a powerful, redistributive fiscal union," Mr Crabb told MPs.
"For all of that there is a concerted effort underway to use the pandemic to weaken the bonds of our United Kingdom."
"Where exactly is Welsh Labour when it comes to the union? There was a time when Welsh Labour was a force for unionism.
"But when I hear Welsh Labour politicians talk down, devalue the efforts of the United Kingdom, when I hear them wanting to stop UK government actually spending money on projects in Wales, when I hear them cynically stoke up the rhetoric around the English-Welsh border, and when I hear them criticise and delegitimise visits to Wales of the UK prime minister - I fear that they've turned their backs on the union and thrown their lot in with the nationalists."
Another former Conservative Welsh secretary, Alun Cairns, praised the announcement that UK ministers would decide how a levelling up fund was spent on some regeneration projects in Wales.
The Vale of Glamorgan MP said: "The strength of the union coming to support areas that have been left behind.
"In my constituency there is a desperate need for a railway station in St Athan and a marina in Barry as part of my regeneration plans and ambitions.
"Until now there has not been the means of the UK Government to step in - I say to the secretary of state I want these two projects to be top of his spending list."
But the announcement was criticised by opposition MPs.
Plaid Cymru Westminster leader Liz Saville Roberts said: "That Westminster can single handedly fix regional inequality is fantasy politics.
"It is Westminster which has tolerated, facilitated and at times marshalled the creation of systematic and structural regional inequalities that are the worst of any comparable developed country."
"Through this levelling up fund, Westminster is overruling Wales, sidelining our democracy and dictating our future," she added.
On Wednesday, Welsh Secretary Simon Hart said the levelling up fund was "not about politicians and power - this is about jobs, livelihoods and post-Covid recovery".
"I do wish Welsh Government would stop fretting about their own little status in Cardiff and actually look at the bigger picture," he said.
'Pitting nations and regions against each other'
Responding on Thursday, Ms Saville Roberts said: "The secretary of state's comments yesterday evening that we should stop fretting about what he termed our little status ignores his own significance at the cabinet table or that Wales will lose a fifth of its representation in Westminster at the next Parliament."
"Decisions affecting Wales should be made in Wales by a government that is accountable to the people of Wales."
Meanwhile, the Labour MP for Cardiff North Anna McMorrin said the UK Government's "attempt to control Wales's levelling up fund is riding roughshod over devolution, pitting nations and regions against each other".
"Our Welsh Labour government continues to provide the most generous support packages to business and fully funding free school meals right through to Easter 2022," she added.
'Appalling record' on spending
In a joint written statement issued later, Welsh Government ministers Jeremy Miles and Rebecca Evans said Wednesday's Treasury announcement "would probably represent little more than £50m each year for Welsh projects - a fraction of the funding we have lost as a result of no longer having access to the [EU] Structural Funds".
"Moreover, in contrast to 'the usual way' Barnett consequentials are allocated, none of this money is ring-fenced to Wales," they said.
The Labour ministers accused the Conservative UK Government of having "an appalling record on providing Wales with even a fair share of UK spending, let alone the kind of funding needed to 'level up'".