Confirming the news, the Women's Tennis Association (WTA) said that the Czech player died peacefully, surrounded by her family.
"After a long battle with cancer, Jana died peacefully, surrounded by her family in her native Czech Republic, aged 49," The Guardian quoted Novotna, as saying.
Novotna, who won 24 singles titles in her career, had lost in the Wimbledon final in 1993 and 1997 before becoming the oldest first-time Grand Slam champion in the Open era by beating Nathalie Tauziat in the 1998 final of the third major of the season.
Novotna, who also made it to the finals of the Australian Open once as well as the last-four of the French Open and US Open twice, also enjoyed prolific success in doubles as she lifted 12 Grand Slam titles.
Paying homage to Novotna, WTA chief executive officer Steve Simon insisted that the Czech tennis star was an inspiration for many - both on and off the court.
"Jana was an inspiration both on and off court to anyone who had the opportunity to know her. Her star will always shine brightly in the history of the WTA. Our condolences and our thoughts are with Jana's family," Simon said.
Novotna, who also bagged four Grand Slam titles in mixed doubles, was inducted into the Tennis Hall of Fame in 2005.