The loss of the trio is a hammer blow to the Wallabies' preparations as they enter the second clash at Docklands Stadium trailing 1-0 in the three-match series.
Hard-running Australia centre McCabe and full-back Barnes were among the three Wallabies players stretchered off during their bruising 23-21 defeat in the first test in Brisbane last Saturday.
McCabe had suffered a neck injury similar to the one he picked up during the Wallabies' northern hemisphere tour last year, while winger Ioane would possibly require surgery after sustaining "internal" damage to his right shoulder, the team said in a statement on Monday.
The pair would be joined on the sidelines for the Melbourne test by Barnes, who came straight off the ground at Lang Park after suffering a heavy clash with a team mate.
"Barnes will not be considered for Saturday night's second test, although it is hoped he will have recovered in time to be considered for the third game in Sydney," the statement added.
The injury blows were announced hours after revelations that winger Ioane had failed to attend a court appearance in Melbourne to face an assault charge relating to an incident in March.
In better news for the Wallabies, inside centre Christian Leali'ifano, who was knocked out cold in the first minute of his Wallabies debut at Lang Park, had recovered well and was likely to be available to play the Melbourne test.
"(Leali'ifano) passed the concussion test on the night, so he was actually keen to come back on, but the time had expired," a team spokesman said.
Outside centre Adam Ashley-Cooper, who underwent scans on his shoulder after coming off injured during the Brisbane test, was also likely to be available.
Flanker George Smith, a veteran of 110 tests and the 2001 British and Irish Lions series, was called into the Wallabies squad with uncapped fullback Jesse Mogg and Queensland Reds centre Ben Tapuai as injury cover on Sunday.
No further replacements have been announced by the Wallabies camp in the wake of the injuries.