There is speculation the date in May is being considered by the UK government.
A spokesman for Labour's First Minister Carwyn Jones claimed it would limit the ability of pro-EU parties to have a "genuine referendum strategy".
Plaid Cymru peer Lord Elis-Thomas broke ranks with his party on Sunday by saying the move could boost turnout.
His party leader Leanne Wood, along with Mr Jones, has previously said the referendum should not clash with the Welsh assembly and Scottish Parliament elections.
The First Minister's spokesman said on Monday: "Our position on this is clear - an in-out EU referendum should be held on a different date from that for the assembly elections.
"The Prime Minister will be cutting off his nose to spite his face if he pushed for the votes on the same day.
"Labour, the Lib Dems, and the nationalists in Wales and Scotland stand ready to play an energetic part in a 'yes' campaign for a reformed EU.
"However, having the vote on the same day as the Welsh and Scottish elections will limit our ability to have a genuine referendum strategy.
"We know the Tories are divided on the EU, and so won't deliver a credible campaign."
The Conservatives promised in their general election manifesto to hold an in-out referendum on EU membership by 2017, but ministers have played down talk that it could be held before then.

David Cameron was answering a question in the House of Commons from Democratic Unionist Party MP Nigel Dodds.
Mr Dodds said legacy funding provided in the Fresh Start deal could help relieve financial pressures on police.
He also said no-one should be allowed to rewrite the history of the Troubles.
Mr Cameron said he would consider Mr Dodds's arguments, adding: "We need to make sure the policing of Northern Ireland continues to be properly resourced, not least because we continue to face a terrorist threat today."
The government has set aside a total of Â£150m for bodies that are to investigate Troubles-related cases.
But a failure by Stormont politicians to agree on how best to deal with the past has meant the money is on hold.
Earlier this month, the Northern Ireland Office said no more cash would be made available to deal with police investigations into crimes of the past.

10 February 2016 Last updated at 14:11 GMT
A team at the University of Sussex put heart monitors on horses and showed them pictures of faces to see how they reacted to different emotions.
They found that the angry faces made the horses' heart rates speed up quicker than the happy faces.
Amy Smith, one of the researchers, explains more.

Such investments should be registered in the same way as securities sales, according to the Securities and Exchange Commission (SEC).
It follows a rise in the number of Initial Coin Offerings, or ICOs, through which crypto-currency start-ups have sought quick financial backing.
However, there are concerns that ICOs are creating an investment bubble.
More than $1bn has been raised so far this year in such transactions but there are questions over how investors are to be protected should a venture backed with digital coins fail.
Now the SEC has said that issuers of "distributed ledger or blockchain technology-based securities" must register offers and sales.
"The innovative technology behind these virtual transactions does not exempt securities offerings and trading platforms from the regulatory framework designed to protect investors and the integrity of the markets," said Stephanie Avakian, the SEC enforcement division's co-director.
It was sometimes difficult to see how a firm had justified a large evaluation for its ICO, said Eitan Jankelewitz, a technology specialist and partner at law firm Sheridans.
"If you consider the size of the investment compared to the type of investment that a professional would make, they look disproportionally large," he told the BBC.
He also pointed out that regulators don't need to rely on legislation specifically dealing with crypto-currencies to enforce rules on securities and investments.
"Those sorts of regulations tend to be more principles-based," he explained.
"If a new technology comes along that allows a new activity to be undertaken, then the law would still apply."
The SEC made its comments on ICOs in a report on a crypto-currency investment fund known as the DAO.
Digital coins worth $60m ($41m) at the time were stolen after the DAO was hacked last year.
While the SEC has not decided to make civil charges as a result of its investigation into what happened, it is using the case to caution industry and the market.
A referendum on UK membership of the European Union should not be held on the same day as the assembly elections in 2016, the Welsh government has said.
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The prime minister has said he will "look carefully" at requests for money set aside for dealing with legacy cases to be released to Northern Ireland's police force.
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Horses can tell if people are happy or angry, according to a new experiment.
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A US regulator has said firms looking for backing via crypto-currencies should be more strictly regulated.