Dunne was forced to withdraw from training this morning with a sore Achilles.
The former Manchester City player has been troubled by the problem in recent weeks and Villa could ill-afford to be without his experience for the Wembley meeting with Carlo Ancelotti's side.
However, midfielder James Milner has resumed training after being rested for the game at Bolton last week, also with an Achilles problem.
Fitness race: Richard Dunne (right) has been one of Aston Villa's star performers
O'Neill said: 'Dunne has had a bit of a setback and he came out of training this morning.
'Richard was a bit sore. It is an injury we have to be nursing through. It has been troublesome for a while but he definitely will want to play.'
Dunne has formed an effective partnership with James Collins since his £6million move from Eastlands during the summer transfer window.
Midfielder James Milner has resumed training after being rested for the game at Bolton last week, also with an Achilles problem, and is expected to be fit.
The club's joint £12million record signing was rested for last week's 1-0 away win over Bolton.
Milner, like Dunne, has been a hugely influential figure during the current campaign and is a strong contender to be part of Fabio Capello's World Cup.
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