The development comes after Sky Blues coach Graham Arnold said he intended to name Antonis in his squad for the Allianz Stadium match, after the star midfielder was ruled out of Australia's international against Japan due to a head injury he sustained driving to the airport on Sunday.
"FFA would like to clarify that in accordance with the FFA Statutes, players selected for international duty who are subsequently withdrawn due to injury or illness cannot play for their club during the period for which the player should have been released," an FFA statement read.
"Therefore, FFA confirms that Terry Antonis is ineligible to play for Sydney FC against Melbourne Victory this Saturday."
Arnold had flagged naming Antonis, while deferring final authority to chief executive Tony Pignata.
"As far as we're concerned he'll be named in the squad later this week," Arnold said.
Antonis suffered minor head and neck injuries in his car accident but was cleared of any serious damage.
Socceroos medical staff and coach Ange Postecoglou decided it was not in Antonis's best interests to fly to Japan and he was replaced in the national squad by Swiss-based midfielder Oliver Bozanic.
Sydney FC has since monitored Antonis closely in line with the FFA's six-day concussion policy.
Arnold appeared confident of playing Antonis, saying he would complete a partial training session with teammates on Thursday and a tactics session on Friday.
Pignata had also weighed in, tweeting on Wednesday evening that Antonis would be picked to play if passed as medically fit. On Thursday he made his feelings known:
The FFA ruling does little to help Arnold's already-depleted side. Strikers Marc Janko and Corey Gameiro are on international duty and captain Alex Brosque was unable to finish Thursday's training session because of an ankle injury.
Melbourne Victory is at least as badly off, with up to nine players unavailable through injury or international duty including skipper Mark Milligan, Kosta Barbarouses, Daniel Georgievski, Connor Pain, Scott Galloway and Jason Geria.
But that's cold comfort for Arnold.
"You saw what they did against Wellington with a so-called depleted squad," he said in reference to the Victory's 2-0 win earlier this month.
"People are tipping them as championship material and that's the depth of the squad they've got."