Villa have the best defensive record in the Barclays Premier League - conceding just 18 goals in 23 games - and O'Neill said: "Statistically it is very good that we have such a good defensive record at the present time.
"I'm aware that could be eroded in two or three games and the defence will be put to a severe test in the next two games against Tottenham and Manchester United, never mind the FA Cup match.
"I've been very pleased and I think James Collins, Richard Dunne and Stephen Warnock coming into the football club in August were incredibly important for us because Curtis Davies was going to be out injured for four or five months."
While hoping Villa can keep Defoe quiet, O'Neill acknowledges the encounter will inevitably lead to people comparing the ability of Defoe and Agbonlahor as Fabio Capello starts to finalise his plans for England's World Cup squad.
O'Neill added: "Defoe is an exceptional goalscorer who always looks dangerous, while Agbonlahor has added other parts to his game.
"The two goals Gabby scored against Fulham will have heightened his confidence again which I don't think has ever been too low.
"It's great to watch him and he is going along very nicely in the goalscoring charts.
"I think the comparisons and contrasts will inevitably be made, as much to do with the fact that Emile Heskey and Peter Crouch are also involved in this match.
"All are obviously desperate to be on the England plane to South Africa.
"The game is very important and to come out of this run of matches after the League Cup final is over it would be nice to still be in that chasing pack. That is our aim.
"Pressure is there all the time and it is relentless and it never goes away. On the plus side this is what you set out to be involved in.