The Americans defeated Australians Nat Cook and Tamsin Hinchley 21-18, 21-19 in group play. It was the last match of the opening day of competition and did not end until shortly before midnight.
The top two from each of the four-team groups will earn a spot in the knockout round, which will take place during the second week of the London Olympics.
May-Treanor and Walsh became one of the top American stories of the Beijing Olympics as they swept to the gold medal. They are seeded only third in this year's tournament, but the top two seeded teams both lost their opening match Saturday.
No. 1 seeds Larissa Franca and Julian Silva of Brazil fell in two sets to Germany's Katrin Holtwick and Ilka Semmler.
The No. 2 seeded team of Xi Zhang and Chen Xue from China also lost in three sets to Russia's Anastasia Vasina and Anna Vozakova.
In the men's bracket, Americans Sean Rosenthal and Jacob Gibb needed only two sets to defeat South African's Freedom Chiya and Grant Goldschmidt. Rosenthal and Gibb are seeded fourth.
Celebrity Families of 2014 [PHOTOS]