Iyer (105) Gill (104) put on a dominant second-wicket stand of 200 to lay the foundations for India's 399-5 after being invited to bat in Indore.
Stand-in-skipper KL Rahul's 52 and an unbeaten 72 by T20 sensation Suryakumar Yadav contributed to the mammoth total and the bowlers then combined to hand India an unbeatable lead in the three-match series.
The matches are a tune-up for the World Cup in India starting October 5.
Captain Rohit Sharma and star batsman Virat Kohli will return for the final ODI in Rajkot on Wednesday and the team will have to make some tough calls in picking the starting XI.
"It (selection) is the headache of the coach and Rohit...whoever gets picked has a job to do," said stand-in skipper Rahul.
"Sitting out can be hard after scoring a lot of runs but everyone has been through that."
Chasing a DLS revised target of 317 in 33 overs after rain interrupted play in the chase, Australia were bowled out for 217 in 28.2 overs.
Spinners Ravichandran Ashwin and Ravindra Jadeja took three wickets each.
Aussies' losing streak
Australia have lost five ODI matches in a row but skipper Steve Smith remained hopeful of a quick turnaround.
"We have lost quite a few (matches) in a row, in South Africa and here. We need to sort out a few things," said Smith.
"We have a few days now, we are working towards the World Cup, both the teams are. Hopefully we'll turn it around in the last game."
Indian batsmen set up victory after a flying start and Iyer, 28, put doubts over his fitness to rest, hitting his third one-day century before being caught.
Iyer hit 11 fours and three sixes in his 90-ball knock and was named man of the match.
The 24-year-old Gill, who hit 74 off 63 balls at Mohali in the first game, raced to his sixth ODI century and fifth this year with a single off Sean Abbott.
Gill's knock ended when he was caught by wicketkeeper Alex Carey off Cameron Green. He hit six fours and four sixes in his 97-ball innings.
Ishan Kishan was dismissed for 31 before Rahul and Yadav propped up the innings with fluent knocks.
Green took two wickets but gave away 103 runs off his 10 overs.
In reply, Australia lost two wickets for just nine runs and were 56-2 when rain interrupted play for over an hour and the tourists resumed with a new target.
David Warner and Marnus Labuschagne attempted to rebuild in their stand of 80 but the wheels came off the chase after their departure.
Ashwin broke the stand and then struck twice in one over to send back the left-handed Warner and Josh Inglis.
Green attempted to hit back but was run out, before Abbott led an unexpected charge with his 36-ball 54.
Number eight Abbott put on a ninth-wicket stand of 77 with Josh Hazlewood, who made 23.
