MacKenzie, a member of the victorious U.S. Curtis Cup team, was ousted by Jennie Lee, 4 & 3, in the first of two match play rounds contested on Thursday at Pumpkin Ridge Golf Club's Witch Hollow Course.
It was a disappointing finish to what MacKenzie, 23, said would be her final amateur appearance.
'There's sadness, a little bit, but at the same time I made a good showing,' said Mackenzie, who earned medalist honors Tuesday after finishing two rounds of stroke play at three-under.
'I'm looking forward to the future,' she said. 'It's sad any time there's a transition. but I'm looking forward to (LPGA) qualifying school.'
Only two previous USGA champions remained in the field Thursday, and both were eliminated along with MacKenzie in the second round of match play.
In Kyung Kim of Korea, the 2005 U.S. Girls' Junior winner, was bested by Eileen Vargas of Colombia, 3 & 2, while Jennie Arseneault upended reigning U.S. Women's Amateur Public Links champion Tiffany Joh in 19 holes.
Joh was trying to become just the second person to win the Women's PubLinx and the Women's Amateur in the same year.
In the third round of match play, Lee advanced again with a 2 & 1 victory over Sydnee Michaels.
The other two spoilers -- Vargas and Arseneault -- also moved on, Vargas with a 1-up victory over Mina Harigae and Arseneault with a 2-up win against Carlota Ciganda of Spain.
The other second-round winners were Ayaka Kaneko, Stacy Lewis, Lindy Duncan, Kimberly Kim and Germany's Katharina Schallenberg.
There are two more days of match play before a champion is crowned following the 36-hole match play final on Sunday.