Moukoko broke the previous record of Leverkusen's Florian Wirtz, who was 17 years, 34 days when he scored against Bayern Munich in June.
Berlin, Germany - His night to remember was spoilt by a 2-1 defeat to Union Berlin, however.
Borussia Dortmund's Bundesliga title aspirations suffered a fresh dent on Friday as they crashed to a 2-1 defeat at Union Berlin on the night their striker Youssoufa Moukoko became the league's youngest ever goalscorer.
After Liverpool loanee Taiwo Awoniyi headed Union into the lead early in the second half, Moukoko leapt into the air to celebrate making history when he equalised for Dortmund aged just 16 years and 28 days.
Moukoko broke the previous record of Leverkusen's Florian Wirtz, who was 17 years, 34 days when he scored against Bayern Munich in June.
However, Moukoko's delight was short-lived as Dortmund again conceded from a set-piece with Union centre-back Marvin Friedrich heading the winner from a corner with 12 minutes left.
"We have now lost two league games through set-pieces -- that's a disaster," Dortmund defender Mats Hummels fumed to DAZN, also referring to November's 2-1 shock home defeat to Cologne.
"We're simply beating ourselves up, that can't be allowed to happen."
Saturday's 5-1 thrashing at home to Stuttgart cost head coach Lucien Favre his job, but Dortmund's defensive woes were exposed again by Union, who also beat them 3-1 in eastern Berlin last season.
Despite bouncing back in mid-week with a 2-1 win at Werder Bremen under caretaker coach Edin Terzic, Dortmund were again punished for yielding possession easily and sloppy defending.
This is Dortmund's fifth defeat this season, leaving them fourth in the table, six points behind Leverkusen who host Bayern Munich in Saturday's top-of-the-table clash.
Union, who held European champions Bayern to a 1-1 draw in their previous home game, climb to fifth, just a point behind Dortmund.
They deservedly took the lead behind closed doors when Awoniyi slipped his marker Giovanni Reyna and headed past Dortmund goalkeeper Roman Buerki on 57 minutes.
"The fact that an opposing striker was so free is unforgivable," Hummels complained after the final whistle.
Having previously hit the Union woodwork, Moukoko made history three minutes later when he stabbed home the equaliser, but Friedrich was left alone in the area to head the winning goal in off the post.
"It's great that we win the game through two set-pieces," Union captain Christopher Trimmel told ZDF.
"There was a lot of passion, no opponent has it easy against us."
The Bundesliga starts a two-week winter break after this weekend and Dortmund have a tough month ahead in January when they face RB Leipzig, Bayer Leverkusen and Borussia Moenchengladbach.
However, Moukoko clearly has a bright future after now claiming three records in the last month.
"You have to congratulate the young 16-year-old on his Bundesliga goal, that's something very special," Trimmel added graciously.
Moukoko also became the Champions League's youngest player ten days ago, coming off the bench in the win at Zenit St Petersburg with Dortmund through to the last 16 where they face Sevilla.
He also became the youngest player in Bundesliga history on November 21 -- the day after his 16th birthday.