The Italian had an unblemished 26-out-of-26 record from the spot for his various clubs and his country ahead of the match. That all changed when he came up against former Liverpool goalie Pepe Reina just after the hour mark in the match against Napoli, with Milan trailing 2-0.
Balotelli tried his usual stuttering run-up, but instead of wrong-footing Reina and passing the ball into the net, he succeeded only in prodding it straight into the path of the diving Spanish shot-stopper.
The former Manchester City star later made up for it with a long-range curler into the top corner, but with just a few minutes left by then it was too late for a comeback. Napoli capitalised on the rare penalty miss to run out 2-1 winners at the San Siro.
Elsewhere, AS Roma ended a five match winless run against arch-rivals Lazio with a 2-0 win which maintained their 100 percent start to the season.
Inter now have 10 points from four games and are unbeaten but they trail leaders Napoli and AS Roma who both recorded their fourth straight wins.
Bayern top scorer Mario Mandzukic, Franck Ribery and substitute striker Claudio Pizarro also found the back of the net as the treble-winning Bavarians notched their fifth win in six league games to join Borussia Dortmund on top of the Bundesliga.
They also extended their unbeaten run to 31 games, five short of the all-time Bundesliga record.
Dortmund, who remain league leaders because of a superior goal difference, dropped their first points this season, drawing 1-1 at Nuremberg, after winning their first five games of the campaign.
Bayer Leverkusen continued their stellar run with a 4-1 demolition of hosts Mainz 05 to stay within one point of the leaders, in third place on 15.
Barcelona stayed on top of La Liga by maintaining their perfect start with a 4-0 victory at Rayo Vallecano but Atletico Madrid are right on their heels after a 2-0 win over Real Valladolid made it five wins from five to leave them second on goal difference.
Promoted Villarreal remained unbeaten on their return to the top flight but slipped down to fourth with 11 points, after being held to a 0-0 draw at Celta Vigo.
PSV Eindhoven took over as leaders of the Dutch league after they scored four goals in 15-minutes on the way to a 4-0 win over champions Ajax Amsterdam.
PSV moved two points clear at the head of the standings on 15 and remain the only unbeaten side after seven rounds. Ajax stay on 11 points, amid a cluster of clubs.
Monaco and Paris St Germain battled to a 1-1 draw in the French Ligue 1 clash of the summer's big spenders in Paris.
The highly anticipated confrontation between the Qatari-backed champions and the ambitious Monaco, who have splashed out around 170 million euros (£143.5 million) in transfers this summer, did not prove as spectacular as had been hoped.
Some big names failed to shine, especially Edinson Cavani, the Ligue 1 most expensive player ever after PSG paid 64 million euros (£54 million) to Napoli to land him in the summer.
Olympiakos Piraues dropped points for the first time this season but they stayed on top of the Greek Super League after a goalless draw at Panaitolikos.
Olympiakos lead the standings with 13 points, with PAOK Salonica second, one point adrift following a 1-0 home win over Platias.
Panathinaikos returned to winning ways with a 1-0 home win over OFI Crete thanks to a goal from Swedish striker Marcus Berg, a result which moved the Greens up to fifth place with eight points.
FC Salzburg striker Jonathan Soriano continued his prolific season by scoring again as the Austrian Bundesliga leaders drew 2-2 at home to Wolfsberger AC.
Soriano's 55th minute goal, which put Salzburg 2-0 up after a first-half effort from Senegal striker Sadio Mane, was the Spaniard's 12th in nine league games this season and his 15th in all competitions.
However, the visitors hit back when Michael Liendl and Sandro Gotal scored in the last half hour. Salzburg stayed top with 19 points from nine games, two clear of promoted Groedig who drew 2-2 with Wacker Innsbruck.
FC Basel followed up their Champions League win at Chelsea by recovering from an early shock to beat FC Sion 3-1 away.
Brazilian Leo Itaperuna gave Sion a 23rd minute lead before Basel hit back through Valentin Stocker, Giovanni Sio and Marco Streller.
Basel pulled level at the top with Young Boys, who lost 1-0 at home to FC Lucerne, and Grasshoppers, who beat FC Thun 2-1.