The announcement was made during a news conference at the museum, which was built around the Lorraine Motel in Memphis, the place where Martin Luther King Jr. was assassinated in 1968.
Rusesabagina, who managed a Belgian hotel in the Rwandan capital, Kigali, whenbroke out in 1994, is receiving the International Freedom Award.
As thousands were massacred throughout the country, Rusesabagina hid 1,268 people in the hotel to keep them safe from Hutu extremist militias and soldiers who killed more than 500,000 minority Tutsis and politically moderate Hutus.
The story of Rusesabagina, a Hutu, and his wife Tatiana, a Tutsi, is the basis of the 2004 film "Hotel Rwanda" - for which actor Don Cheadle, who portrayed Rusesabagina, was nominated for an Academy Award.
"The world turned a deaf ear and closed its eyes to what was happening in Rwanda," said National Civil Rights Museum director Beverly Robertson. "But he stepped up, even at the potential expense of his own life."
Winfrey - who runs a media company that includes her television shows and a magazine - has been active in promoting many causes including, most recently, aid for tsunami victims and children in South Africa.
Robertson described Winfrey "a great humanitarian" who has used her Oprah Winfrey Foundation to provide scholarships and grants to help educate poor children in the United States and abroad.
"She has provided resources and money to build schools for at least 50,000 children in South Africa... That is phenomenal. She doesn't have to do that," said Robertson, also praising Winfrey for her advocacy of the 1993 National Child Protection Act.
The museum also announced a new Lifetime Achievement Award for civil rights activism for actressand her late husband, .
Dee and Davis made their film acting debuts in 1950 in "No Way Out" with Sidney Poitier and performed together on Broadway in "A Raisin in the Sun." They were friends with King and longtime activists for civil rights causes.
Previous recipients of the Freedom Awards include King's widow, Coretta Scott King, former South African President Nelson Mandela, Georgia Rep. John Lewis, former Presidents Bill Clinton and Jimmy Carter, and Bono, the Irish rock star and human rights activist.
The museum gives out Freedom Awards annually to individuals who have worked to advance civil rights.
The award winners will be honored at a banquet in November.