The mistake happened when Wolfsburg's cup tie at fourth-tier minnows Preussen Muenster on August 8th went to extra-time, during which Van Bommel made a sixth substitution. Competition rules allow only five changes.
It was an embarrassing mistake for the ex-Bayern Munich midfielder during his first competitive game in charge of the Wolves, who have qualified for the Champions League.
Wolfsburg won 3-1 at Muenster after extra time, but the hosts appealed to the German Football Association (DFB) when they realised the visitors' mistake.
As a result, Munster's name will go into the draw on August 29th for the second round after the DFB disqualified Wolfsburg on Monday night, and awarded Muenster a 2-0 win, following a five-hour hearing.
"This is the most bitter decision you can make. We had no room for manoeuvre," said Stephan Oberholz, deputy chairman of the DFB sports court in Frankfurt.
Wolfsburg can appeal the decision and are looking at their legal options.
"We are disappointed and would have preferred a different outcome. We do not agree with the decision and will now examine legal action," said Wolfsburg's managing director Dr. Tim Schumacher.