Michal Bilek's side complicated their World Cup hopes after drawing against Bulgaria and losing to Denmark in their last two home games, with a 3-0 win in Armenia in March at least keeping them third in Group B.
Friday's match in Prague is expected to go ahead despite floods that have left swathes of the country submerged and threatened parts of the capital's historic centre.
"We will try to raise the spirit of the people by beating Italy," goalkeeper Petr Cech told a Czech newspaper.
Euro 2012 runners-up Italy lead the group with 13 points from five games, ahead of Bulgaria on 10 from six and the Czech Republic with eight points from five matches with five more to play.
The top side qualify directly for Brazil 2014 with the runners-up facing a play-off.
"It will not be easy but it all depends on us," Cech added. "A victory would put us back in the running for qualification for Brazil but one point would also keep us in the hunt. A loss would make things extremely complicated."
One factor for the Czech team will be whether playmaker Tomas Rosicky can put in a full 90 minutes. The diminutive midfielder spent much of the year injured but came on strong at the end of the season for his English club side Arsenal.
Rosicky's skills will mean nothing if the Czechs continue to struggle in front of goal with only six goals scored in their five matches so far.
Italy, who have no real surprises in their squad, have knocked in 12 goals in their five matches but are taking nothing for granted in Prague before they head off to the Confederations Cup in Brazil.
"The Czechs are one of those teams you have to go out and beat because they don't give you anything," goalkeeper Gianluigi Buffon told reporters.
"You play a below-par game then you risk losing, if you play only normally then you might be able to grab a point but we want to play a great game to increase our lead at the top of the group."
Get the best features, fun and footballing frolics straight to your inbox every week.
Thank you for signing up to Four Four Two. You will receive a verification email shortly.
There was a problem. Please refresh the page and try again.