The former Inter Milan striker stepped off the bench to stun Timisoara and hand his side the advantage going into the return game next Thursday at Eastlands.
Balotelli, 20, proved to be a handful and has already repaid the first part of his reported £24million transfer fee.
After a slow start on a humid night, England's biggest spenders were good value for the victory thanks to a positive second-half performance.
Urged on by a large partisan crowd, Timisoara opened with confidence and sprayed the ball about well.
The home fans screamed for a penalty after 14 minutes when Alexandru Curtean went to ground following a challenge from Pablo Zabaleta.
German referee Florian Mayer ignored their pleas and had no hesitation in waving play on.
City, with a small pocket of supporters backing them, were taking their time getting into the game. Carlos Tevez was dropping deep on occasions, leaving Emmanuel Adebayor up front on his own.
Roberto Mancini's side then won a free-kick 25 yards out in the 30th minute after David Silva was tripped by Alexandru Bourceanu.
Tevez stepped up and tried to float his effort beyond Costel Pantilimon but the goalkeeper saved at the post.
Play switched back down the other end and Cosmin Contra cracked in a shot from an acute angle that was deflected to safety.
City, however, were beginning to look dangerous and David Silva picked out Tevez at the back post.
He tried to chest the ball into the path of Adebayor but his pass was intercepted and the move broke down.
Srgian Luchin became the first player to be booked with a stiff challenge on Tevez that left the striker hobbling.
Tevez dusted himself down and picked out Gareth Barry a minute before half-time with the midfielder lurking on the edge of the area.
He was about to shoot only for Luchin to show good positioning and take the ball off his toes.
City were on the front foot at the start of the second half and Adebayor found Zabaleta on the edge of the area. He tried his luck on goal only for his effort to finish nearer the corner flag.
Back came Timisoara and Dan Alexa slipped a glorious through ball to Mircea Axente in the 51st minute.
The striker whipped in a shot but Joe Hart got down quickly to make a comfortable save.
Mancini decided to switch things around and handed Balotelli his debut in the 57th minute when the 20-year-old replaced Gareth Barry.
The Italian's arrival improved City's attacking options as they went in search of an important away goal.
Silva had put in a hard shift and was taken off in the 66th minute with Adam Johnson coming on.
City were beginning to put the pressure on and Balotelli found the side netting a minute later after a misplaced overhead kick by Tevez.
Then Yaya Toure's thunderbolt came back off the post following a corner from Balotelli as Timisoara began to buckle.
City made the breakthrough with Balotelli showing a cool head to sweep the ball home from close range after being set up by Adebayor.
It was just the boost City were looking for with the second leg to come next Thursday at Eastlands and they are now favourites to progress to the group stages.