The move is one of three changes in the Australia backline announced by coach Ewen McKenzie, who has made six changes in total.
O'Connor has been replaced in the starting side by centre Tevita Kuridrani, who will take over the number 13 jersey, with Ashley-Cooper switching to the wing.
ACT Brumbies flyer Joe Tomane has also been included on the other wing in the place of Nick Cummins.
"Adam has great versatility, he is our most experienced backline player," McKenzie told reporters on Thursday.
"To be honest, we put numbers on their back but it doesn't mean they will play like that. We have a physically imposing backline and we hope to put pressure on the Boks with that. Tevita Kuridrani has also been itching for a chance."
Australia captain James Horwill returns from injury to take over from Kane Douglas at lock, the only other change to the starting line-up from the side that beat Argentina 14-13 last time out.
"James Horwill coming back is logical as captain. We have got the best guys that are available now and they are improving as a combination."
New Zealand-born wing Chris Feauai-Sautia, who plays for Queensland Reds, has been added the bench and will make his international debut if the 19-year-old gets onto the pitch.
Waratahs loose-head prop Benn Robinson has also been added to the bench to replace Scott Sio.
McKenzie acknowledges that his side were well short of their best when thumped 38-12 by South Africa in Brisbane earlier this month, but is comfortable with the underdog role for this match.
"The one game we were disappointed with was the game against the Boks because we did not feel we got close to how we wanted to play, in the forwards or the backs.
"All we have read about since we have been here is how much the Springboks are going to beat us by... we have taken note of that."
Team: 15-Israel Folau, 14-Adam Ashley-Cooper, 13-Tevita Kuridrani, 12-Christian Leali'ifano, 11-Joe Tomane, 10-Quade Cooper, 9-Nic White, 8-Ben Mowen, 7-Michael Hooper, 6-Scott Fardy, 5-James Horwill (captain), 4-Rob Simmons, 3-Ben Alexander, 2-Stephen Moore, 1-James Slipper
Replacements: 16-Saia Fainga'a, 17-Benn Robinson, 18-Sekope Kepu, 19-Sitaleki Timani, 20-Ben McCalman, 21-Will Genia, 22-Matt Toomua, 23-Chris Feauai-Sautia