In the lawsuit, a copy of which was obtained by The Associated Press, Wade is seeking at least $50,000 in damages from each defendant.
Siohvaughn Wade said last month the player infected her with an unspecified disease in 2007. Weeks later, she withdrew the claim from the court hearing the divorce case for unknown reasons.
That wasn't enough to satisfy the 2006 NBA finals MVP, who said her allegations not only defamed him, but caused "embarrassment" and "humiliation" along with damaging future earning potential.
"It has been difficult to see false allegations, rumors and gossip repeatedly discussed in public about my personal life," Dwyane Wade said in a statement released to the AP. "I had no other choice but to file this action in order to clear my name."
Wade declined further comment.
The All-Star guard also lists attorneys Michael Berger and Dorene Marcus as defendants, along with their firms, contending they wronged him in the same manner as his estranged wife. The suit was filed in Chicago, where the divorce proceedings have gone on for nearly a year.
"Mr. Wade has been the victim of a reckless campaign by Siohvaughn Wade and her lawyers to publicly humiliate him and damage his reputation," Joe Power, an attorney representing the Heat player, said in a statement to the AP. "To clear the public record from these false and malicious allegations, we have filed a defamation suit in court ... and will seek appropriate compensation for damages."
Siohvaughn Wade's claim made "by and through her attorneys, and disseminated by various print and online media throughout the world placed plaintiff Dwyane Wade in a false light," according to the lawsuit.
"We believe there's absolutely no merit to it," Berger said. "I believe that the lawsuit will get dismissed. It seems interesting to me Wade would file a lawsuit like this when he supposedly wants to keep a low profile. This lawsuit will put his entire private and social life in full view of the public."
Berger added that even though the original claim by Siohvaughn Wade was withdrawn, "Mrs. Wade has stood behind her allegations."
In the allegations filed in January, along with the claims of affairs and being infected with an STD, Siohvaughn Wade said her husband abandoned their children and goes weeks at a time without speaking to them. She added that the paternal relationship was so severed that her younger son cries uncontrollably when he's around and he does not recognize Dwyane Wade as his father.
Siohvaughn Wade's withdrawal of the claim wouldn't prevent her from resubmitting it to court at a later date. The divorce proceedings are not expected to end anytime soon.
Dwyane Wade's private life has been tabloid fodder for several months, a span that largely coincides with his split from his high school sweetheart.
A recent story in The Palm Beach Post, based on comments made by a former business partner of Dwyane Wade -- a person who's filed suit against him, alleging breach of contract in a failed restaurant deal -- said Wade used a rented Miami apartment for sex parties and smoked marijuana.
Wade's representatives denied that story, dismissing it as lies from someone seeking a payoff from the NBA star.
Dwyane and Siohvaughn Wade began dating as teens and continued their relationship through college. He starred at Marquette, leading his team to the Final Four before being drafted with the No. 5 overall pick in 2003 by Miami.
The couple married in 2002 and have two children, the older of whom was born three months before the wedding. Their second son was born in 2007.
Through the tumult, Wade has been having the best season of his career in many statistical areas. He entered play Wednesday as the NBA's second-leading scorer.
"He's done a tremendous job just focusing on the team and the game when he's in this building," Heat coach Erik Spoelstra said. "In many ways, this can be a sanctuary, not just a place of work, but a place of escape and he's got a great way of compartmentalizing things in his life."