Eldor Shomurodov gave Uzbekistan an early lead but Newcastle United forward Yoane Wissa dragged Congo back level with a converted penalty on 68 minutes. Fiston Mayele then put DR Congo ahead on 78 before Wissa struck again in stoppage time to seal a historic win.
Wissa, who scored three goals across the group stage, was overwhelmed by the scale of the achievement in his post-match comments.
"I'm just one piece of the puzzle," Wissa said. "Today was very tough — the weight on our shoulders was hard to bear. There are things we need to fix.
"But the most important thing is this historic Round of 32. We're going to savour this moment because it's been tough.
"All the guys — the substitutes, those who've worn the jersey before, and those who'll wear it tomorrow — we should be proud. Thank you to all the Congolese people; it's for moments like these that we do what we do. We did it!"
Head coach Sébastien Desabre paid tribute to his players' ability to fight back when it mattered most.
"We have to congratulate the players — they were the ones who were extraordinary," Desabre said. "We're a team that knows how to respond when we concede a goal; we keep fighting with determination. We were a little more offensive today, and I'm glad everyone could see that we're capable of scoring goals. That's great. I have to congratulate my coaching staff as well; we're really proud and happy for all Congolese people."
DR Congo's qualification ends a wait that stretches back to 1974, the only previous occasion the country appeared at the World Cup finals. For Uzbekistan, a debut campaign full of fight and ambition ends at the group stage despite an impressive showing across the tournament.