"Following the tragedy in Orlando, Broadway performers came together to perform a song in support of victims and their families," Rudolph said before the song began.
They then launched into a heartfelt rendition of "What the World Needs Now Is Love." The song is available online with proceeds going to benefit the LGBT Community Center of Central Florida.
In the early hours of June 12, Omar Saddiqui Mateen entered the Pulse nightclub in Orlando, which caters to a gay clientele, and opened fire, killing 49 people and wounding 53 more.
Authorities told ABC News that Mateen had been "on the radar" of U.S. terrorism agencies but was not the subject of any specific investigation.
The mass killing took place just one day after another tragic shooting in Orlando, when gunman Kevin Loibl shot and killed 22-year-old singer and former "The Voice" contestant Christina Grimmie.
Following the shooting, the U.S. Senate took up four gun control proposals after a 14-hour filibuster by Sen. Chris Murphy. However, all of the proposals failed on a party line vote.
Watch the video above.