Foot soreness sidelined Healy from Monday night's match, where Australia lost the toss and were sent in, chasing a win that would ensure they retain the urn.
Healy was sidelined during last year's T20 World Cup when she injured the same foot that caused her pain after Australia's clean sweep of the Ashes One Day Internationals.
Gardner, player of the match in the most recent ODI, has been diagnosed with a low grade calf strain and will be assessed ahead of the second of three T20Is.
On T20I debut, Georgia Voll partners Beth Mooney at the top of the batting order in Healy's place, while Grace Harris slots into the middle order to replace Gardner.
Voll excelled as Healy's replacement when a knee injury rubbed the captain out of the recent ODI series against India, averaging 86.5 with the bat across her three innings.
Harris, meanwhile, plays her first international match since the shock loss to South Africa in the semi-final of last year's T20 World Cup.
Mooney is keeping wicket in Healy's absence, having done so during recent series against India and  New Zealand, while vice-captain Tahlia McGrath becomes skipper.
The leading-wicket taker in the multi-format Ashes series, Alana King will play her first T20I since February 2023.
King has been overlooked for T20Is in recent times but a knee injury to fellow spinner Sophie Molineux cleared a spot for her in the bowling attack alongside front-line tweaker Georgia Wareham.
Cricket Australia is still assessing whether Healy will return for the final two T20Is or one-off Test match of the Ashes series.
"Cricket Australia's medical team is consulting with specialists to develop an appropriate management plan over the coming days," a statement read.
"Her availability for the remainder of the series will be assessed as more information becomes available."
Fighting to keep their series alive, England have brought in Sophia Dunkley, Freya Kemp and Sarah Glenn to the XI that lost the third ODI in Hobart.
Tammy Beaumont drops from the top of the batting order, with quick Lauren Filer and allrounder Alice Capsey also omitted.
AUSTRALIA
Georgia Voll, Beth Mooney, Phoebe Litchfield, Ellyse Perry, Annabel Sutherland, Tahlia McGrath (c), Grace Harris, Georgia Wareham, Alana King, Kim Garth, Megan Schutt
ENGLAND
Maia Bouchier, Danielle Wyatt-Hodge, Sophia Dunkley, Nat Sciver-Brunt, Heather Knight (c), Amy Jones, Freya Kemp, Charlie Dean, Sophie Ecclestone, Sarah Glenn, Lauren Bell
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