The world number three, leading 4-0 in the first set, slipped on the Hisense Arena surface then turned her right ankle and lay motionless on the bright blue court before sitting up when medical attention arrived.
The reigning Wimbledon and US Open champion received treatment courtside, which included having her ankle re-strapped, and returned eight minutes later.
The 31-year-old appeared to be favouring her ankle and grimaced from time to time, but had obviously decided to limit her movement and blasted winners seemingly at will in order to get off court as quickly as possible.
There is no bigger personality in tennis than Williams who is chasing a third successive Grand Slam title after wins at Wimbledon and the US Open.
"It was definitely a lot of pain," she said. "Also a little bit of the memory, as well. So it was definitely a little bit of both. But also at the same time trying to gather myself together and trying to make sure that I can continue."
She said nothing would stop her from taking to court for her second round tie against Spain's Garbine Muguruza.
"I'll be out there," she said. "I mean, unless something fatal happens to me, there's no way I'm not going to be competing. I'm alive. My heart's beating. I'll be fine."
World number one Victoria Azarenka got her title defence underway with an unconvincing 6-1 6-4 victory over Romania's Monica Niculescu on Rod Laver Arena.
The Belarusian did well to battle back to secure victory without going to a third set after going 3-0 down in the second, but the way her first serve fell apart at times will only encourage her main rivals for the crown.
Second seed Maria Sharapova got her campaign off to a blistering start without losing a game on Monday before Williams followed suit.
Azarenka will perhaps reassure herself that she will be more battle-hardened after her 87-minute win over Niculescu as she prepares for a second-round tie against Eleni Daniilidou.
Former world number one Caroline Wozniacki came from behind to grind out a 2-6 6-3 6-3 victory over tough first-round opponent Sabine Lisicki.
The 10th seeded Dane later said she felt that Lisicki should have been seeded, such was the quality of the German's play.
Petra Kvitova was also taken to three sets by an unseeded player, the Czech eventually beating Italy's Francesca Schiavone 6-4 2-6 6-2 to book her place in the second round.
Seventh seed Sara Errani was bounced out at the first time of asking as the Italian fell to a 6-4 6-4 defeat to Spain's Carla Suarez Navarro.
Victory for the Spaniard ranked as the biggest shock of the tournament so far as Errani became the second seed to tumble out on the day.
Earlier, Japan's Kimiko Date-Krumm also produced a shock when she unseated 12th seed Nadia Petrova 6-2 6-0 to become the oldest winner of a women's singles match at the tournament.
The 42-year-old, playing her 11th Australian Open 23 years after her debut, was well worth the quick-fire victory, which she sealed on her second match point when her Russian opponent hit a forehand wide.
Date-Krumm reached the semi-finals at Melbourne Park in 1994, where she lost to Steffi Graff, but had not won a match at the year's first Grand Slam since returning after a gap of 12 years in 2009.
The world number 100 will face Shahar Peer of Israel or another Russian in Alexandra Panova in the second round.