Market researcher Ipsos Mori found 58% of respondents said their firms had suffered since last June's exit vote.
Nearly a third of respondents thought the referendum had not made any difference, and 11% felt it had been positive.
More than 100 bosses of FTSE 500 firms responded to the survey.
"Unfortunately, it looks like business in this country is already feeling the pain of the economic upheaval of leaving the EU," said Ipsos Mori chief executive, Ben Page.
"According to respondents there is no sign that this is likely to ease this year, with two thirds saying they thought their business situation would get worse in the next 12 months," he added.
Two-thirds of the chairmen, chief executives and other directors who responded thought that their business would be worse off after the UK left the European Union.
Meanwhile 45% of the 114 FTSE 500 bosses surveyed expected to feel a negative impact in five years' time.
