The 46-year-old showed some vintage touches in an entertaining 5-7, 7-6, 6-4 victory over American duo Lisa Raymond and Mike Bryan.
Earlier in the day the American, who debuted at the All England Club in 1976, chalked up her 300th match at Wimbledon when she and Russian partner Svetlana Kuznetsova were beaten 4-6, 6-2, 6-4 by Belgium's Kim Clijsters and Ai Sugiyama of Japan in the women's doubles quarter-finals.
Navratilova has nine singles crowns, seven women's doubles and three mixed doubles successes to her name to date.