The Orioles promoted Mancini to the major leagues on September 18, 2016 during September call-ups. On September 20, in his major league debut as the designated hitter against the Boston Red Sox, he hit a home run against Eduardo Rodríguez in his second at bat, making Mancini the fourth Orioles' player to hit a home run for their first Major League hit after Larry HaneyNick Markakis and Jonathan Schoop. Mancini started again two nights later and hit his second career home run, a three-run homer off David Price. He became the 20th player in Major League history to hit a home run in his first two starts. He would then become the third player in Major League history to homer in their first three starts, after he hit a solo home run against the Arizona Diamondbacks on September 24.
2017
Mancini made the Orioles' 2017 Opening Day roster after transitioning to the outfield. On April 16, Mancini recorded his second multi-home run game of the season, while driving in four runs. He tied Trevor Story and Dino Restelli for most home runs through a players first 12 career games with seven. On April 22, Mancini hit his fifth home run of the season and eighth of his career. He tied Story and Carlos Delgado for most home runs through a player's first 17 career games. On June 7, in a game against the Pirates, Mancini hit a game-tying, pinch-hit two-run home run in the ninth inning off closer Tony Watson. Two innings later, with two on and two outs, Mancini hit a walk-off three-run home run off Wade LeBlanc to give the Orioles a 9-6 victory. Per STATS LLC, Mancini became the first Oriole to hit a pinch-hit homer in the 9th inning or later and then hit another home run in the same game. He also became the second Oriole ever to hit a game-tying home run in the 9th inning or later and then hit a walk-off homer in the same game. He also joined Boog Powell and Eddie Murray as the only three Orioles to homer in the 9th inning and in extras in the same game. On June 25, Mancini hit his 14th home run of the year, giving him the most home runs before an All-Star break by an Orioles rookie.
2018
Mancini batted leadoff for 51 of 156 games played in 2018. He ended the season hitting.242 with 24 home runs and 58 RBI.
2019
Mancini got off to a strong start in 2019 and was leading the Orioles in most offensive categories in late-April. He is one of three MLB players along with Alex Bregman and Anthony Rendon with at least 35 doubles, 34 home runs and 100 runs in. He set a career high with five hits in an 11-4 win over the Toronto Blue Jays at Rogers Centre on September 24, 2019. He ended the season hitting.291 with 35 home runs and 97 RBI.
2020
On March 7, Mancini left the Orioles during spring training to undergo a non-baseball medical procedure. On March 12, it was revealed that the procedure was the removal of a malignant tumor from his colon. On April 28, Mancini revealed he had stage 3 colon cancer and will begin chemotherapy ongoing for six months to treat his cancer. Mancini said if baseball resumed in 2020, it would probably be without him.