Lambert quashed speculation that the club were in discussions to sign the Belgium international and also ruled out another move for Nurnberg midfielder Hiroshi Kiyotake.
Defour has been linked to Villa by his agent during the past month, but Lambert claims he will not be making an offer to Porto, while a move for Kiyotake is not on the cards, despite Villa's tentative interest in the summer.
When quizzed on the speculation regarding Defour, Lambert told the Birmingham Mail: "No, there's nothing in that. I've not seen that one."
Lambert provided a similar response when Kiyotake, who Villa enquired about last summer before deciding against pursuing the Japan international, was mentioned.
"Who? At Nuremberg?" Lambert questioned. "It was a lad I tried to get in the summer, but things have moved on from that. Nuremberg are fighting to stay in the Bundesliga. Good player."
Lambert is looking to strengthen his midfield ranks, but was staying coy on rumours he wants to sign Wes Hoolahan from his former club Norwich.
The Villa boss refused to discuss Hoolahan and insists Villa have alternative creative midfielders in mind, as well as potential signings in other positions.
"There's no point talking about somebody else's player," he added. "You look at other things and other options and if you can do it you look to do it.
"There are a few No.10s I looked at, as I call them, and some we just can't do. There's other positions I'd like to strengthen. We're trying to bring in a couple.
"If we can do it, then great, but this window is so difficult to deal with. You just can't bring in people for the sake of it. It really is a tough window to do anything."