The People's Elephant defeated AS Real de Bamako 1-0 in the second leg of the first round inside the Stade Modibo Keita in Bamako after losing the first leg a week ago in Aba by 1-2.
Enyimba head coach, Zachary Baraje, dropped first-choice goalkeeper, Femi Thomas for the game and handed veteran shot-stopper, Chijioke Ejiogu, a rare starting place against the Malian club.
Despite Zaka Bala's goal just before the end of the first half of the match on the day, it was AS Real de Bamako that progress on the away goal rule.
It is only the second time that Enyimba will fail to reach the group stage of the Caf Champions League in their eighth appearance.
In 2002, Ivorian club, ASEC Mimosas stopped Enyimba in the second round following a 5-4 win on aggregate.
But Enyimba went on to win the competition back-to-back in 2003 and 2004.
The ouster of the six-time Nigerian champions now means that no club from the West African nation will take further part in this year's edition of the Caf Champions League.
Defending champions of the Nigerian premier division, Kano Pillars, were bundled out of the same competition over two legs by DR Congo's AS Vita Club in the preliminary round last month.