Libor Kozak had put the visitors ahead in Turin after 19 minutes, sparking fears among home fans that what had looked set to be a celebratory night could take a turn for the worse.
However, the Azzurri scored twice in the space of four minutes - first Giorgio Chiellini, then Balotelli - to secure a spot in Brazil next summer.
Gianluigi Buffon equalled Fabio Cannavaro’s all-time record of 136 caps as took his place in goal, while there were recalls for Balotelli and Riccardo Montolivo following their suspensions.
Michal Bilek made three changes to the side that suffered a shock defeat to Armenia on Friday.
Marek Suchy, Vaclav Prochazka and Vladimir Darida came in for Michal Kadlec, Josef Husbauer and Michael Rabusic.
Despite the hosts having the better of the opening exchanges, it was the visitors who broke the deadlock as Kozak fired home in the 19th minute following a swift counterattack.
The Aston Villa striker escaped the attentions of the Italian defence to meet Petr Jiracek’s cross and guide the ball past Buffon.
Italy should have equalised six minutes later but Balotelli missed two fantastic chances from inside the penalty area.
His first struck the crossbar after a good strike from Emmanuele Giaccherini’s cross, and he blazed his second over the bar after Cech had spilled a shot from the edge of the box.
All the home side's chances were falling to the Milan striker but he could not find a way past Cech.
When Andrea Pirlo floated a perfect cross towards the 23-year-old, he could only head straight at the Czech goalkeeper, who pushed the ball away.
Cesare Prandelli's side levelled six minutes into the second half through Chiellini. Cech missed his punch from a Pirlo corner and the Juventus defender rose unmarked to head into the empty goal.
And on their next attack they moved ahead as Balotelli was brought down in the penalty area by Gebre Selassie.
He picked himself up to calmly fire past Cech, sending the Chelsea goalkeeper the wrong way.
Italy had their visitors firmly under control and when substitute Daniel Kolar received a second yellow card for a poor challenge on Balotelli, the party was able to start.
They saw out the closing moments of the match comfortably to secure top place and seal qualification with two games remaining.