The former Presidents also took to the stage to deliver messages to the audience.
Following her performance, Gaga again returned to Instagram to share photos from backstage, where she met with the former Presidents.
"It was an honor to be invited by these five living Presidents to perform and speak at this historic event where we put our differences aside, and put humanity first in the face of catastrophe," she captioned a group photo.
Presidents Carter, Clinton, Obama and Bush Sr. and Jr. started the One America Appeal in the wake of Hurricane Harvey, which struck Texas in September.
They also assisted in the wake of Hurricanes Irma and Maria.
As of Saturday night, the charity had raised US$31 million.
Lady Gaga made a surprise appearance at the benefit, which also featured performances by Lyle Lovett, Robert Earl Keen, Sam Moore and Yolanda Adams.
Taking to Instagram before the event, Gaga shared a series of stunning photos of herself backstage and ready to go.
"One America Appeal," she captioned them, giving each photo a single word caption.
"Nothing more beautiful than everyone putting their differences aside to help humanity in the face of catastrophe. #OneAmericaAppeal," she also tweeted.
After the show, a photo shows her standing in between a blue curtain and red floral arrangements.
"What's a girl to do, but red white and blue?" she captioned the patriotic photo.
Meanwhile, a third photo showed her flashing a peace sign at the camera.
"Thank you for establishing a mental health and trauma recovery fund within #OneAmericaAppeal which I donated to personally for Hurricane Relief," she captioned it.
"The @btwfoundation is in awe of this commitment to rebuilding these communities emotionally and psychologically."
