Deloitte said "unqualified" EU support among financial bosses at leading UK companies dropped to 62% from 74% although 'a clear majority of CFOs continue to favour membership.'
Some 28% stated they were awaiting the outcome of the Prime Minister's renegotiation with European leaders before making up their mind and just 6% felt UK businesses would benefit from leaving the union, although this was up from 2% initially.
Another poll, by the Financial Times (FT), found that not one of more than 100 leading economists felt a Brexit would be beneficial for the country's economic prospects in 2016.
The figures come ahead of an inout referendum which could be called as early as this summer, and has been promised by David Cameron before the end of 2017.
David Cameron's renegotiation is entering a crucial phase ahead of a key EU summit in February where a deal could be reached.
Deloitte asked 137 chief financial officers (CFOs), including 24 from FTSE 100 companies, whether it was in the interests of UK businesses for the country to remain in the EU.
The main swings in opinion since the last survey six months ago were a greater uncertainty and a narrower margin for the "In" camp, the auditors said.
Some 4% of CFOs were "uncertain", compared with 1% previously.
David Sproul, senior partner and chief executive of Deloitte, said: "A clear majority of CFOs continue to favour the UK remaining in the EU, but the proportion of those expressing unqualified support has fallen. This mirrors what we have seen from the broader public in opinion polls in the last six months.
"With around one third of CFOs undecided on their position or awaiting the outcome of renegotiation discussions, the eventual deal may well significantly affect business attitudes to EU membership."
The FT poll also found 76% of economists felt the UK economy would be worse off in the medium term as the result of a Brexit, while only 8% thought prospects would be better.
Adam Posen, president of the Institute for International Economics, told the paper a vote to leave the EU "changes my views about 2016 and the medium term drastically for the worse".
"Business investment will dry up," he added.
Carolyn Fairbairn, director general of the Confederation of British Industry (CBI), said business' uncertainty was "understandable".
She told the BBC's Today programme: "We have a negotiation going on by the Prime Minister and what businesses are telling me is they are waiting to see the deal. That's entirely understandable.
"The proportion of business leaders who want to be out under any circumstances is still very low."
Our BQ Bulletin emails will land in your inbox at 7.30am, Monday to Friday, with a mix of the latest local business news, national news, and features to inspire you. Sign up here!
Click here to read our privacy statement