It was Ronaldo's third goal in two games and rewarded the Portugal squad for the relentless pressure it had applied all through the match.
The Czechs did not have a shot on goal, and only two attempts during the match. Portugal, the Euro 2004 runners-up, had 20 attempts.
Portugal advances to play Spain or France in the semifinals in Donetsk, Ukraine, on Wednesday.
In Warsaw, there were no taunting chants of "Messi, Messi, Messi" directed at Ronaldo as there had been at past matches, but the crowd whistled derisively when the Real Madrid star touched the ball.
Messi has claimed three straight Ballon d'Or trophies since Ronaldo won the award in 2008, and the Argentine's Barcelona side ruled the Spanish league for years until Real Madrid finally took the title this year. Even then, Ronaldo finished second to Messi in goal-scoring.
And then there were Ronaldo's national team scoring woes. Over an 18-match period, he scored only twice - once from the penalty spot - earning the scorn of many Portugal fans and a reputation for failing to shine on soccer's biggest stages.
At Euro 2012, Ronaldo looked as if he were headed down that same road, with lusterless - and scoreless - performances in Portugal's first two matches against Germany and Denmark.
Then came his two-goal game against the Netherlands, and now the winner against the Czechs.
At the final whistle, Ronaldo dropped to his knees with clenched hands, then rose as Miguel Veloso jumped into his arms in celebration.