Madrid still have the advantage of a game in hand over their closest rivals, but Zidane has called on his players to expect teams to be fired up to face them.
"It's getting more difficult," he told a news conference. "They're a very good opponent, well organised defensively. It's good for us to play [on Sunday], after the defeat on Wednesday, so we can try to put it behind us.
"It was an opportunity that we let slip, but you shouldn't keep coming back to it, it's not something that can be fixed any more. We just have to think about the next game. What happened, happened.
"We know that we can and should improve, always. For the teams who play against Real Madrid, it's the game of the year. And that's normal, it's an extra motivation, it's something we have to take into account."
3 - Real Madrid have lost more games in 2017 (3) than they had last year (2 - all competitions). Alarm. pic.twitter.com/K57s6ZwxwK— OptaJose (@OptaJose) February 22, 2017
Barca's clash with Atletico could give Madrid a boost in terms of their advantage at the top, but Zidane is refusing to focus on anything other than beating Villarreal.
"We can't control that game. We're only looking at ours. The only thing we can control is what we do on the pitch," the coach said.
"Until the end of the season, nobody knows what will happen. We had a big disadvantage last year and at the end we had a chance. You never know what will happen in football."
Sergio Ramos rejected the assertion that Madrid were punished for a poor attitude at Mestalla and Zidane backed his captain's comments.
"There was no lack of attitude," he said. "We had two mistakes that cost us two goals, but the attitude was very good. You have to analyse and think about what happened and be more focused in key moments.
"It's difficult to maintain concentration thinking that the opposition can score at any moment."
Gareth Bale has been tipped to return to the starting line-up at Estadio de la Ceramica, with Zidane confirming that he has recovered full fitness since his return to training following an ankle injury.
He also suggested that Marco Asensio will be given some time on the pitch, having sat out the last three league matches.
"Apart from [Raphael] Varane and Danilo, everyone is okay," the former midfielder explained. "Bale has had a few days training with us, he's at 100 per cent.
"I'm very happy with Asensio's work. But everyone knows that, when there are only 18 places in the squad, someone has to be left out.
"I'm happy with Asensio; he'll get some minutes."
Madrid confirmed this week that Varane has suffered a hamstring strain, and although he does not think the injury is too serious, Zidane acknowledged that the centre-back's absence is a big blow.
"It's a very tough loss," he added. "He's played a lot of matches, he's a very important player, as he has shown with his form. He's out for an indefinite period.
"With a grade two injury, it's normally not too long, but there are other players who are ready to play in his position. We're not happy with the injury but there's nothing we can do."