Udinese's penalty shootout defeat to Braga, a side made up largely of journeymen Brazilian players, on Tuesday means Italy will have only two teams in the group stage.
After falling to Arsenal at the same stage last year, Udinese's latest setback has also raised worries about the future of the team who have habitually overachieved in recent seasons and been one of the success stories in the league.
Coach Francesco Guidolin, who led them to a third-place finish last season, fourth the season before that and saw his best players sold off on both occasions, did not think he would get another chance to reach the group stage with the club.
"Evidently I am not capable of leading a team into the Champions League," said a dejected Guidolin, who has been persuaded to stay on.
"When you get so close several times and can't go through, you have to learn from that experience and accept the truth."
Before the match, he had warned that failure could be the end of Udinese's impressive run.
"It cannot be taken for granted that this team can carry on doing so well as, to do that, we would have to be magicians and I cannot perform miracles."
Udinese must now try and pick themselves up and face titleholders Juventus at home on Sunday.
Their defeat has led to soul-searching in the Italian media although, rather than looking for solutions at home, some are suggesting that it is time for UEFA to merge the Champions League and Europa League into one huge tournament.
Italy had its quota of automatic Champions League places cut from three to two this season after Serie A dropped below the Bundesliga in the rankings used to calculate the number of berths per country.
Udinese's misfortune came on top of a miserable summer marked by the Calcioscommesse match-fixing scandal, which led to points deductions for four Serie A teams, and the failure to attract top names to the country.
If Italy needed a timely reminder of why the top players no longer want to come, then the confusion over the venue for Sunday's Cagliari-Atalanta match has provided it.
The Sardinian side has moved out of Stade Sant'Elia, its home since 1970, due to disagreements with the local authorities and growing safety worries.
During the second half of last season, these forced Cagliari to play home games in Trieste, 1,000 kilometres and closer to Belgrade than their own base.
Cagliari had intended to move to the Is Arenas stadium in the commune of Quartu Sant'Elena, around five kilometres outside the city.
But with workmen rushing to finish necessary improvements to the new arena, Cagliari said in a statement that permission to play the match had not been granted.
With 72 hours to kick-off and faced with the option of another trip to Trieste, Cagliari were still trying to convince the league that the new venue would be ready.
- Champions League