Kohli made an eight-ball duck as India posted an underwhelming 9-136 from 26 overs in the rain-affected series opener at Optus Stadium.
Australia cruised to the victory target with 29 balls to spare, with skipper Mitch Marsh (46no off 52 balls) starring on his home deck.
Kohli is officially retired from Test and T20 international cricket, meaning ODIs are now the only time he will turn out for India.
The soon-to-be 37-year-old is aiming to make it to the 2027 ODI World Cup in Africa, but whether his form will hold up for that long remains to be seen.
Kohli's appearance in Perth was his first match since guiding Royal Challengers Bengaluru to the IPL title in early June.
His most recent appearance for India came in an ODI against New Zealand in March.
Kohli is likely touring Australia for the final time as an international player, and he only has games in Adelaide on Thursday and in Sydney on Saturday to ensure his Down Under farewell is a memorable one.
Indian paceman Arshdeep Singh has no doubt Kohli will fire this series.
"I mean, he's played more than 300 one-dayers for India, so form is just a word for him," Singh said.
"He knows how to get going, and it's like a blessing to be in the same dressing room with him.
"Going forward, I feel there'll be a lot of runs for him in this series as well.
"And talking about the format he's playing, he's mastered this one, so I don't know how he feels about it (the 2027 World Cup). I'll ask him about his feelings and maybe tell you in the next press conference."
Australia can wrap up the three-match series with victory in Adelaide.
Spinner Matt Kuhnemann snared 2-26 from four overs while bowling at the death, but he is likely to be squeezed out for game two with Adam Zampa (paternity reasons) set to be available.
Kuhnemann is accustomed to playing second fiddle, having only featured in five ODIs, five Tests, and one T20I so far in his career.
The 29-year-old says he will accept any selection decision on the chin.
"I'm used to it," Kuhnemann said.
"Being able to travel with the red ball team and running drinks there, and sort of just learning my way, I love travelling with this team, and you never know when conditions might suit two spinners.
"I'm always happy to be part of this team and learning and growing as a player."
Kuhnemann is expecting India to rebound strongly in Adelaide following their disappointing display in Perth in front of 42,423 fans.
"I think they'll come back really strongly," Kuhnemann said.
"They're a world-class team, and it's going to be a great series."
Marnus Labuschagne, who was a late inclusion in the squad following Cameron Green's side niggle, is a chance to earn a spot in the XI in Adelaide.
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