CBI Wales chair Chris Sutton said the proposed vote in 2017 was creating "uncertainty".
David Cameron pledged to renegotiate a "better deal" for the UK and hold an "in-out" referendum by the end of 2017.
Chancellor George Osborne said the UK would be "constructive but resolute" in talks with EU leaders.
Mr Sutton said: "From a business perspective the EU isn't perfect but I think business sees it as a real positive - it's a single market of 500 million people and I think it will be difficult for jobs and growth for us to step out of that."
He added that the business organisation would be campaigning to stay within a reformed EU and warned that firms would not like the uncertainly in the two years leading up to the referendum.
"The departure from the EU is not a revolving door; if we get it wrong we can't just come back in again. There would be huge amount of renegotiating of positions in terms of bilateral trade agreements and an impact on our national wealth for us to come out of it."
BUSINESS REACTION - YES OR NO TO A REFERENDUM?
Bethany Sawyer is general manager of Biopharm UK - based in Hendy in Carmarthenshire - which has been supplying leeches for medical uses since 1812.
She believes the two years before a referendum will give time for the UK to negotiate a better EU.
But for Peter Lewis, managing director of engineering firm IAC in Newport, the referendum would cause stability and be a waste of money.
He employs 75 people and is worried a vote to pull out of the EU could be a setback when manufacturing needs a boost.
Many multi-national companies including Tata, Airbus, Toyota and Ford operate in Wales and support a number of smaller suppliers.
They are responsible for tens of thousands of jobs.
Airbus though has said the UK leaving the EU would not result in it relocating.
The UK car industry and the manufacturers' organisation, EEF, has expressed concerns about the prospect of leaving the EU.
West Wales and the Valleys has received over Â£4bn of European funding with another Â£2bn to come over the next seven years to support economic growth and create jobs.
But critics argue that this is nothing compared to the money the UK sends to the EU and so more funds would be available in the event of leaving.
The EU is a major trading partner with Wales.
Elgan Morgan, of the South Wales Chamber of Commerce, said: "Trading with EU countries is the first step for many businesses when they are looking to export their goods and services.
"Leaving the EU would complicate relationships between Welsh businesses and their European customers and may discourage businesses from pursing export possibilities altogether. This must be avoided at all costs."
New Conservative MP for Gower Byron Davies said the referendum was something that they had canvassed on and people agreed with holding it.
Plans to hold a referendum on EU membership are unhelpful for businesses in Wales and could lead to reduced investment, warns an industry body.