The group, which includes Virgin founder Richard Branson, say that Brexit would damage Britain's economy.
Smaller businesses and employers would be particularly vulnerable to any economic shock as a result of a Leave vote, according to its signatories, who include the leaders of 50 of the FTSE 100 companies.
"Britain leaving the EU would mean uncertainty for our firms, less trade with Europe and fewer jobs," they say in the letter to the Times newspaper.
The letter goes on to claim that the terms of trade between Britain and its European market would have to be reset "from scratch" in the event of a Brexit.
Elsewhere, in another eve-of-poll move, the UK Prime Minister David Cameron said, "If we stay in the EU, that is not the end of reform. Britain will be in a very enhanced position. There is no doubt that a British Prime Minister with a mandate from the election, a re-negotiation, and a referendum would have the ability in Europe to drive forward changes that are needed."
A further warning about the impact of a Brexit comes from Polish Europe Minister Konrad Szymanski.
He told the Financial Times, "Any significant, post-Brexit, deal with Europe would need to address the problem of free flow of labour force and proper guarantees for migrant workers. We will prioritise it during possible future negotiations…Brexit is a bad scenario for Europe and for those who want to reform the Union to restore its principal roles."
He added, "Disintegration of the EU means weaker Europe and weaker member states in the world."
The Belgian government, meanwhile, has set up a team of experts to investigate the possible consequences of a Brexit and to assess what Belgium could do to limit the damage.
Belgian economy minster Kris Peeters said that Belgium will be among the EU countries feeling the heaviest impact of a Brexit.
His comments were backed by the Flemish Minister-President Geert Bourgeois, who claimed a Brexit would hit Flanders harder than other Belgian regions.
He said that if the UK decides to leave the EU this could impact negatively on the Flemish GDP, to the tune of 2.5 per cent by 2030.
"However, Flanders is ready to bear the consequences", Bourgeois told Belgian broadcaster VRT.
Two polls released on Wednesday suggested that Remain has regained some ground, while a third one found Leave ahead.
A telephone poll by ORB for the Daily Telegraph puts Remain on 53 per cent (+5) and Leave on 46 per cent (-3). The poll finds that turnout among Remain voters has risen nine points in the last week to 69 per cent.