The victory over the 80th-ranked Kovinic, which is just her second this year, will be a confidence boost for Williams but the path to a record-equalling 24th Grand Slam now gets treacherous.
Waiting in the wings is Estonian second seed Anett Kontaveit, who breezed past Jaqueline Cristian 6-3 6-0.
Williams indicated her intention to retire in a Vogue article in early August, saying she was "evolving away from tennis" but never confirming the US Open as her final event.
For tennis fans, however, the message was clear, the US Open would be where she would take her final bow.
Could there be an encore?
The former world number one has played coy even refusing to rule out next year's Australia Open.
But in a strange post-match ceremony celebrating her career, which was not yet finished, Williams gave the clearest sign yet that the US Open will indeed be her last tournament and expanded on that later in her press conference.
"It's extremely difficult still because I absolutely love being out there," said Williams. "The more tournaments I play, I feel like the more I can belong out there.
"But it's time for me, you know, to evolve to the next thing."
Pressed if the US Open was definitely her last event Williams again stepped back from the brink.
"I've been pretty vague about it, right," smiled Williams. "I'm going to stay vague because you never know."
When Williams leaves the stage she will not be walking away from the sport she says has given her "everything".
"I love the sport and I feel like it's given me so much," said a reflective Williams. "It's given me a platform that I never even thought I would have.
"I don't see myself not a part of tennis.
"I don't know how I'm going to be a part of tennis as of right now. I just don't know how.
"But I just feel like we've come too far together to just not have anything to do with it."
While Williams has dominated the women's game for over two decades, she also has many other interests.
She has been a fashion designer, an actress and has backed early-stage companies. She was an early investor in MasterClass - one of 16 unicorns, companies whose market value exceeds $1 billion, to receive funding from Serena Ventures.
While there are certain to be more business opportunities to grow out of tennis, Williams' greatest impact and connection to the sport is likely to continue to be as a leader speaking out on diversity, racism and other social issues.
Williams has been unable to define what her future tennis role might be, but the 32-time Grand Slam winner is aware of the impact she has had off the court but never dwelt on it.
"I don't think I've even taken a moment to realize any impact," said Williams. "I understand it, but I don't really meditate or think about it.
"I'll have plenty of time soon to do all that.
"I never thought I would have that impact, ever.
"I was just a girl trying to play tennis in a time where I could develop this impact and be a voice."
A montage of Williams' career played before she appeared on court that left the door open a crack for a return someday, "if you ever decide to return Queen your throne will be waiting" ended the video.
A defiant Williams, however, made it clear she was not giving up that throne just yet.
SPECIAL ENERGY
There is always excitement on opening night at Flushing Meadows but on August 29th the teeming stadium crackled with a special energy from the moment Williams appeared on court decked out in a shimmering black robe and specially designed diamond-encrusted shoes.
The outfit may have sparkled more than the American's play but it did not matter to a jam-packed Arthur Ashe Stadium as Williams' fighting edge remained razor sharp even if her serve and groundstrokes were not.
"It’s so important to give your all," Williams told the adoring crowd. "I’ve been down and out so many times, in the public eye.
"I just want people to be inspired by my story.
"I’m from Compton, California… and I made it."
Certainly, there was no more fitting place to bring the curtain down on one of tennis's most phenomenal careers than in a city that has been in her corner from the very beginning, fuelling runs to six US Open crowns.
The magnitude of the moment was not lost on the 23,000 fans, including, former US president Bill Clinton, designer Vera Wang, director Spike Lee and others who packed into Arthur Ashe not expecting to see greatness but to celebrate it.
Having been world number one for 319 weeks, Williams arrived in New York ranked below 600 and unseeded.
Even with Williams far from her best the odds were stacked against the 27-year-old from Montenegro.
Playing in her 21st US Open, Williams has never lost in the first round and her victory over Kovinic was her 106th at Flushing Meadows.
While Kovinic has been enjoying the best Grand Slam season of her career reaching the third round of both the Australian and French Open, she had not won a single match since Roland Garros.
As play got underway it was Williams showing signs of nerves, piling up the double faults as Kovinic got in front 3-2.
But Williams, as she has done so many times, lifted her game when she needed to, sweeping the next four games to snatch the first set.
Now in charge, Williams would not let it slip away in the second set, breaking to go up 3-2, and with match point and the crowd on its feet, she danced in delight as Kovinic's return hit the net.
