The collision on October 28 near the town of Urda, 130km south of Madrid, left almost 200 others injured.
The Foreign Affairs Department initially flagged the two Australians had potentially been affected on October 30 and confirmed they had been killed on Wednesday.
"The Department of Foreign Affairs and Trade is providing consular assistance to the families of two Australians who died in Spain," a department spokesperson said in a statement.
"We send our deepest condolences to the families at this difficult time."
The victims have not been named for privacy reasons.
More than 500 people were aboard when a train operated by state-owned rail company Renfe crashed into the back of a stationary train operated by Iryo, a private company.
On Wednesday, local authorities confirmed the nationalities of all 69 killed, including 54 Spanish citizens.
No further victims are expected to be found.
It's the second deadly railway accident this year after another train crash in Andalusia, in Spain's south, killed 46 people in January.
Spanish authorities are under pressure to ascertain the cause of the crash after audio recordings of interactions between train drivers and a control centre were published by the media.
The recordings confirmed the control centre had ordered the Renfe train to resume its journey while all other trains on the line were kept stationary due to a faulty safety system.
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