Williams had served for the first set before losing four games in a row and was 4-0 up in the second before she weathered a Tomljanovic fightback to take it on a tie-break.
But Tomljanovic remained composed in a partisan atmosphere and took advantage of a fading Williams to race away with the third set.
Speaking after the match, Tomljanovic told the crowd: “I’m feeling really sorry because I love Serena just as much as you guys do.
“What she’s done for me, for tennis, is incredible. She is the greatest of all time.”
Asked afterwards if she would consider a comeback, Williams said: “I don’t think so, but you never know.”
And she has been true to her word. She will return on the grass courts of West Kensington when she plays doubles alongside 19-year-old Canadian star Victoria Mboko at Queen’s.
Williams’ elder sister Venus still plays on the WTA Tour and reached the quarter-finals of the 2025 US Open women’s doubles, aged 45.
“I don’t think Serena will come back and be satisfied with making the second week,” Tomljanovic said before the American’s return was confirmed.
“Even her last US Open, I think in her mind she was going to win it, so she must be feeling some sort of extra boost of energy.
“Maybe she’s inspired by Venus.”