The 18-time major-winner said Court's statement that homosexuality was a choice suggested she may have wrestled with the issue.
Court's stance against same-sex unions has drawn threats of protests at the Australian Open.
"You say it is a choice to be gay; do you mean to say you had feelings for women as well as men and chose men? That might explain your certainty on the issue," Navratilova wrote in the Herald Sun newspaper.
"You frame the whole gay issue in religious terms and quote the Bible," she added. "While I am not a theologian, I do know these same Bibles have been used in the past to justify slavery, to deny men of colour the right to vote and to try to deny inter-racial couples the right to marry."