Playing in the last tournament of her illustrious carer this fortnight, the six-time Flushing Meadows winner fell in three sets to Tomljanovic. After losing a tight opening set 7-5, Williams restored parity by taking the second in a tiebreak, much to the delight of a record crowd at the Arthur Ashe Stadium.
However, the 23-time Major winner found a 5-1 deficit too big to surmount in the decider. Despite saving five match points, Williams lost in the third round at Flushing Meadows for the first time since making her US Open debut.
At her post-match press conference, Williams, when asked about the proudest moment of her illustrious career, said that her Roland Garros triumph in 2015 would remain her proudest moment, as she 'almost died' during the tournament.
"French Open 2015, yeah. That is definitely the one that I'm taking with me because I almost died in that event. Somehow I won. So that was pretty awesome," Serena Williams said.
In Paris that year, the former World No. 1 won her third title at the tournament, but the win didn't come without drama. Against Lucie Safarova in the final, the then 33-year-old looked on course for a routine win - leading by a set and a break. Suddenly, a double fault would change the course of the match.
Safarova broke back and took the second set on a tiebreak to level proceedings. Trailing in the decider, Williams roared back to reign supreme in Paris for the third time, winning her 20th Major title that year.
Winning the first three Majors that year, Williams was two wins away from a calendar year Grand Slam but lost to Roberta Vinci in one of the biggest Grand Slam upsets in recent history.
"The crowd was really wanting to push me past the line, I'm so thankful and grateful for that" - Serena Williams
Serena Williams was the darling of New York this fortnight even though she fell well short of winning the title.
Fans thronged in huge numbers to catch the legend for one last time before she walked into the sunset and Williams didn't disappoint. Having won just one of four singles matches all year coming into New York, the 40-year-old caught fire.
After seeing off Danka Kovinic in straight sets, she dumped out second seed Anett Kontaveit in the next. However, she failed to get over the line against Tomljanovic, despite a record crowd cheering her all the way through.
Overwhelmed by the outpouring of love and support for her all week, Williams - widely regarded as the best female athlete of all time - said that she's grateful for the same.
"It was some really great moments today. Not today, but this week, has been really great. Just so much support, so much love. It was really amazing and overwhelming. So grateful for it. I am so grateful for it. And I just feel like, you know, the whole crowd was really wanting to push me past the line. I'm so thankful and grateful for that," she said.
Serena Williams' third-round conqueror will next take on Ludmilla Samsonova for a place in the last eight.