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.
Born to an Ethiopian father and a Czech mother, he is the first black player to represent the Czech national team and has made the right back position his own in the last year.
Given his chance after the Czech defence performed badly in a 3-0 defeat by Norway last year, the 25-year-old has not looked back since although he has never played for a club outside his homeland and has been with Slovan Liberec since 2008.
Until then, he had barely played against foreign opponents.
Limbersky has also spent most of his career at home and has been with Viktoria Plzen for the last three years, helping the small provincial club win an improbable title in the 2010/11 season.
The two sides met at Euro 2008 with the Portuguese winning, but the Czechs still enjoy harking back to their famous 1-0 win at Euro 96.
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."
The Czech Republic were thrashed 4-1 by Russia in their opening group game at Euro 2012 but bounced back to qualify with wins over Greece and co-hosts Poland.
However, the Portuguese, whose national dish is dried cod, will be a different kettle of fish.
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."
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