The encounter, played behind closed doors at the TP Mazembe Stadium, saw the Congolese side take a valuable advantage ahead of next Tuesday's return fixture in Nigeria.
DR Congo opened the scoring through an own goal involving Nigeria captain and goalkeeper Samuel James after a controversial incident in the Nigerian penalty area.
The hosts doubled their advantage in the 40th minute with a well-taken free-kick, leaving Nigeria with a two-goal deficit at half-time.
Injury setback for Nigeria
Nigeria suffered another setback around the half-hour mark when Brighton youngster Zadok Yohanna was forced off with an injury.
His withdrawal disrupted the visitors' attacking plans as they struggled to find a response against a determined Congolese side.
The Olympic Eagles improved defensively after the interval and prevented the hosts from extending their lead, but they were unable to find a breakthrough of their own.
Nigeria face must-win return fixture
The defeat leaves Nigeria with a difficult task ahead of the second leg, scheduled for Tuesday, 17 November, at the Remo Stars Stadium in Ikenne.
The Olympic Eagles must overturn the two-goal deficit to keep their hopes of reaching the final qualifying round alive.
The winner of the two-legged encounter will advance to face either Burkina Faso or Sierra Leone in the next round.
The 2027 U-23 AFCON in Egypt will also serve as Africa's qualifying tournament for the men's football competition at the 2028 Olympic Games in Los Angeles.