Adam Taggart got on the scoresheet for Australia but it was not enough as two Australian-born players were in the FYR Macedonia side that defeated the Olyroos 3-1 in Skopje.
The Australians were unable to repeat the heroics of their 1-0 win over Turkey in Istanbul on Friday, as they continue preparations for January's Olympic Games qualifiers.
The winning FYR Macedonia side included Nicola Kuleski and Phil Petreski, who were both raised in Australia.
Kuleski plays for Sydney FC's youth team, while Petreski, another midfielder, was Melbourne City's NYL player of the year last season.
FYR Macedonia went ahead in the third minute from a free kick by Demir Imeri.
Fringe Socceroos striker Taggart levelled the score but the Red Lions struck again before the break, after captain Viktor Angelov found the back of the net.
Winger Petar Petkovski made the game safe shortly after the hour mark, after a mazy run and precise finish.