Matildas captain Sam Kerr has been ruled out of the two matches against Olympic champions Canada due to a foot injury.
The star striker will remain in London for treatment with her place in the squad taken by midfielder Sarah Hunter, the 20-year-old earning her first senior national team call-up.
The news comes a day after it was confirmed Charlotte Grant (hamstring), Holly McNamara (knee), Courtnee Vine (hamstring) and World Cup hero goalkeeper Mackenzie Arnold would also miss the Canada games.
Arnold, who is captain of West Ham United in the English Women's Super League, was ruled out with an arm injury.
Veteran Matildas defender Clare Polkinghorne says the team won't be deterred by the setbacks leading into the friendlies.
"Our main priority is having everyone fit and healthy for the Olympics," Polkinghorne said on Tuesday.
"We would prefer if everyone was available but that's the reality of professional football.
"Injuries are part of the game and unfortunately it's happened the weekend leading into these games (against Canada)."
Australia last took on Canada at the Women's World Cup in July, emerging victorious in Melbourne with a 4-0 thumping which was kickstarted by an electric first-half brace by Hayley Raso.
With Christine Sinclair calling time on her international career, Polkinghorne expects their opponents to come in guns blazing to give the veteran Canadian forward a send-off victory.
"Canada are reigning Olympic champions so there's no letting your hair down against them," Polkinghorne said.
"They're a world-class team and they would not have enjoyed being beaten by us in that fashion (at the World Cup).
"They've probably got a little bit to prove, bit of redemption.
"For us, it's going to be about rising to the occasion and spoiling their party."
Polkinghorne played down concerns about reports linking Matildas coach Tony Gustavsson to the vacant Sweden men's head coach role, adding that Gustavsson had not addressed the issue to the playing group.
Gustavsson, the 50-year-old Swede, is contracted until after Paris 2024 but was also previously heavily linked to the United States women's head coach role, since taken up by Chelsea boss Emma Hayes.
"I wouldn't say it's unsettling. That's part and parcel of professional football," Polkinghorne said.
"If you've had success and done well at the top level, then there's always going to be interest from other parties.
"Whatever is happening outside our circle is not really relevant."
Australia will take on Canada in Langford from 2.30am (AEDT) on Saturday before a second clash on December 6 in Vancouver.
