Right-back Theodor Gebre Selassie, whose international experience was limited to a handful of Champions and Europa League qualifiers until recently, should get the job of stopping Ronaldo tearing down Portugal's left flank.
David Limbersky, who had a brief but unsuccessful spell with Tottenham Hotspur, will try and keep Manchester United winger Nani under wraps as the Czechs seek to repeat their win over Portugal at the same stage of the 1996 tournament.
Gebre Selassie has been one of the Czech's discoveries of the last year but this will be by far his biggest test.
A stunning lob by Karel Poborsky ousted Portugal's so-called golden generation from the tournament and the Czechs went on to reach the final.
"Euro 96 changed my football career and my life in a crucial way, and the same could be said of about seven or eight of my team-mates who were transferred to other clubs after the tournament," Poborsky told UEFA's official website.
"When we saw it on the big screen, we just laughed," he said of his goal. "It was just one moment, a strike of inspiration that cannot be repeated. I know many people remember me only thanks to that goal."
Despite suggestions that he saves his best for Real Madrid, Ronaldo has scored nine goals in Portugal's last 11 competitive internationals and his double in the 2-1 win over the Dutch means he has scored at three successive European Championships.
But the team's passionate coach Paulo Bento is taking nothing for granted.
"We don't claim to be favourites and it won't be easy," he said. "We have to keep up our work and the quality that we have shown so far. Then maybe we can even reach the semi-finals.
"There are still a lot of things to develop but I'm proud, like the players should be."
