Williams had been hampered by illness in the lead-up to the Paris showpiece, but the world number one showed her mettle to hold off a late Safarova fightback and win 6-3 6-7 (2/7) 6-2.
It means Williams, who also won the Australian Open in January, is now only two short of Steffi Graf’s 22 major titles and four away from Margaret Court’s all-time best 24.
While Williams was playing her 24th major final, it was Safarova’s first, and the Czech pushed her world number one opponent to the limit in an enthralling contest.
“It was a very complicated match,” Williams said.
“Lucie was a magnificent opponent for me, she was very aggressive.
“I was a set up and a break and I got nervous but I came through and I so happy to be part of history, winning my 20th grand slam.
“I can’t believe I have won my 20th here in Paris on this court. It is very special.”
All the talk in the build-up to the match had been about Williams’ health after the American spluttered her way through a semi-final victory against Timea Bacsinszky and then skipped practice and press duties on Friday to recuperate in her Parisian apartment.
Williams said she was suffering from severe symptoms of flu and she took a bathroom break before play even started, asking to leave the court for a short time after the knock-up.
Any extra encouragement Safarova had drawn from Williams’ pre-match problems, however, were quickly extinguished as the American announced her arrival with a crashing backhand winner in the opening game.
Safarova had relied on her whipping forehand to beat both Maria Sharapova and Ana Ivanovic on route to the final but Williams gave her no opportunity to unleash as she peppered her opponent’s backhand with power and precision.
In the fourth game, the American struck the first blow as a fearsome forehand return cross-court sealed a break of serve and while Safarova dug deep to hold at 5-2, Williams served out to clinch the first set.
Safarova’s serve was consistent enough but it lacked potency and Williams slapped away a series of thundering returns to break in both the first and fifth games to lead 4-1.
Complacency, or perhaps tension, suddenly took hold as Williams served three double faults to hand one break back, and then another at 4-3 to allow Safarova to pull level.
Williams unleashed another stupendous return to break again and serve for the match at 6-5 but Safarova had now found her rhythm and she hit back immediately to force an unexpected tie-break.
A combination of Williams’ unforced errors and some brilliant defence gave Safarova four set points and she converted at the first opportunity to force a decider.
The momentum was now with Safarova, who was faster in her movement and more confident in her shot-making, and another break fired her into a 2-0 lead at the start of the third.
It was now Safarova’s turn to lose her nerve, however, as a double fault gifted a break back to Williams, who seized the opportunity, winning five games in a row to turn the set on its head.
One game away for a second time, Williams made no mistake as Safarova failed to retrieve a booming forehand and the American wrapped up another grand slam triumph in two hours and one minute.