Agriculture is the biggest item in the commission's budget
The European Commission says several EU governments will have to return a total of 410.3m euros (£326m; $644m) in misspent farm subsidies.
The biggest offender was Italy, which has to give back about 145m euros for not properly checking land areas eligible for EU subsidies.
The commission said it also found poor controls in citrus processing in Italy and evidence of fraud.
Greece has been told to return 127.7m euros and the UK 69.4m euros.
In the Greek case, the commission found inadequate controls in the arable crops and nuts sectors.
In the UK, the commission found poor checking of land areas eligible for subsidies and inappropriate timing of follow-up field visits.
Smaller repayments to the commission are due from France, Germany, the Netherlands, Poland, Spain and Sweden.
Farm subsidies under the EU's Common Agricultural Policy amount to about 49bn euros annually.
The commission says its latest decision is the 28th since the system for recovering misspent CAP funds was reformed in 1995.
Commenting on the decision on Wednesday, EU Agriculture Commissioner Mariann Fischer Boel said: "We are working extremely hard to maintain the best possible control over farm spending".
"The Court of Auditors has noted considerable improvements in our control system over recent years and we are striving to make things better still.
"This is taxpayers' money and they have a right to know it is being spent wisely," she said.