Victory for the Italians would have seen them move onto six points in Group C, but a spirited performance from Czech Republic has thrown the section wide open.
Italy boss Luigi di Biagio made four changes to the side that won 2-0 against Denmark, with Mattia Caldara replaced at the back by Bologna's Alex Ferrari, while Davide Calabria came in for Antonio Barreca at left-back.
Czech Republic's team, meanwhile, showed three changes from the one beaten 2-0 by Germany on Sunday, with Ajax's Vaclav Cerny among those dropping to the bench. Sampdoria's Patrik Schick, however, led the line once again.
It took just 13 seconds for Italy to carve out the game's first half-chance, but after Bernardeschi broke into a dangerous position, there were no takers on the winger's cross into the Czech Republic box.
Bernardeschi also had the first shot on target in the 11th minute after collecting a loose ball inside the Czech Republic box, but the Fiorentina attacker could not find a route past goalkeeper Lukas Zima as Italy passed up a clear opportunity to make the breakthrough.
Czech Republic's first sight of the Italian goal came in the 21st minute when Daniel Holzer found a yard of space just outside the box, but the number 21 sent his effort high of Gianluigi Donnarumma's crossbar.
It was Czech Republic that made the breakthrough in the 24th minute, however, when the skipper Michal Travnik drove to the edge of the Italian box before drilling a low effort just out of Donnarumma's reach.
Berardi came close to levelling less than two minutes later when the Sassuolo forward sent a free kick towards the bottom corner, but the ball just flew wide of Zima's post, before Donnarumma denied Schick with a smart stop down the other end.
Petagna was next to come close for Italy 10 minutes before the interval, but the centre-forward just missed the far post after using his power to wriggle free of a couple of challenges.
Italy made a strong start to the second period as they looked to level the scores, and Berardi forced Zima into a smart save in the 52nd minute, before Federico Chiesa was introduced off the bench two minutes after.
A save from Zima then prevented Bernardeschi's low effort from finding the back of the net in the 56th minute, but the pressure was increasing as the Italians looked to level the scores against a tiring Czech Republic.
Zima was again called into action just past the hour to keep out a free kick from Berardi, but Italy were not to be denied, with Di Biagio's side levelling the scores in the 70th minute courtesy of a close-range header from Berardi, who had seen his initial effort saved by Czech Republic's impressive goalkeeper.
Petagna had a brilliant chance to send Italy ahead 15 minutes from time after breaking into a one-on-one situation with Zima, but the powerful striker somehow lifted the ball over the crossbar.
Czech Republic then made sure of all three points in the 85th minute when Michael Luftner's spectacular long-range effort somehow found a route inside the post to stun the Italians in Group C.