Harry, the younger son of King Charles, co-founded Sentebale in honour of his mother Princess Diana in 2006, nine years after she was killed in a Paris car crash.
Sentebale means "forget-me-not" in the local language of Lesotho in southern Africa.
Co-founder Prince Seeiso of Lesotho, as well as the board of trustees, joined Harry in leaving Sentebale until further notice following a dispute with chair Sophie Chandauka, who has reported the trustees to the United Kingdom's Charity Commission.
"It is devastating that the relationship between the charity's trustees and the chair of the board broke down beyond repair, creating an untenable situation," Harry and Seeiso said in a joint statement published by UK media on Wednesday.
Harry, who lives in the United States with his wife Meghan and two children, stopped working as a member of the royal family in 2020.
He has been involved in charitable causes in Africa for many years and visited Nigeria last year.
The trustees had acted in the best interests of the charity in asking the chair to step down, the joint statement said.
"Although we may no longer be Patrons, we will always be its founders, and we will never forget what this charity is capable of achieving when it is in the right care," it added.
Sentebale said it had not received resignations from its royal patrons.
In a statement, Chandauka said she would continue to perform her role.
"There are people in this world who behave as though they are above the law and mistreat people, and then play the victim card and use the very press they disdain to harm people who have the courage to challenge their conduct," she said.
She added that underlying the "victim narrative and fiction" that she said had been fed to the media was "the story of a woman who dared to blow the whistle about issues of poor governance, weak executive management, abuse of power, bullying, harassment, misogyny, misogynoir - and the cover-up that ensued".
The Charity Commission said it was aware of concerns about Sentebale's governance.
"We are assessing the issues to determine the appropriate regulatory steps," a spokesperson said.
