The world number one was scheduled first on the new Louis Armstrong Stadium against Kaia Kanepi and lasted just an hour and 15 minutes before succumbing to a 6-2 6-4 defeat.
At the other end of the day, Williams opened the night session on Arthur Ashe and proved too strong for Poland's Magda Linette, winning 6-4 6-0.
It was her first match at Flushing Meadows since a semi-final loss to Karolina Pliskova in 2016, having given birth to daughter Olympia during the tournament 12 months ago.
Williams has had a difficult build-up to the tournament, suffering her most one-sided loss ever when she won just a single game against Johanna Konta in San Jose - she subsequently revealed she had learned just before the match that the man who killed her half-sister had been released from prison.
Williams then lost to Petra Kvitova in the second round in Cincinnati but pulled away here after a tight opening to the match and needed just an hour and nine minutes to clinch victory.
The 36-year-old, who is looking to equal Margaret Court's all-time record of 24 slam singles titles, said: "It's such a good feeling to be back out here. It's an experience you can only live in New York and it's one of the best feelings in the world.
"The first set was tight. Once I got settled, I started doing what I'm trying to do in practice, so it helped a lot. I think I'm getting there. I've been feeling really good in practice."