Brentford midfielder Frank Onyeka got Nigeria off to a dream start when he fired in an opening goal in the third minute. However, D.R. Congo equalised in the 31st minute through forward Meschack Elia, who scored just his second goal in World Cup qualifying.
Nigeria's top scorer and talisman Victor Osimhen was forced off through injury in the 46th minute in a massive blow for the Super Eagles. And Nigeria would miss their star striker as they failed to find a winning goal inside 120 minutes, with a tense game going to a nail-biting penalty shootout.
The cagey shootout was edged 4-3 by D.R. Congo, who in turn booked their place in March's inter-confederation playoffs and ended Nigeria's World Cup dream.
Captain Chancel Mbemba converted the decisive kick after Congolese substitute goalkeeper Timothy Fayulu, brought on a minute before the shootout, made two saves in the shootout.
D.R. Congo now await the draw on Thursday for the inter-confederation playoff in March where six teams will chase two places at the 48-team finals.