The second-ranked Clijsters skidded to the net and stumbled on the first point of the second game and appeared tentative for the rest of the match, although she did not call for medical treatment.
Clijsters said she would return home to Belgium and seek medical advice before deciding whether to play at Wimbledon.
"It depends on results from the hospital," she said.
The Belgian initially hurt her ankle dancing at her cousin's wedding in April. The ankle was taped for her brief run at the French Open, where she also lost in the second round.
"It's the same ankle I injured earlier," she said. "I slipped in the first set and it started hurting. I took some medication on the court that helped with pain in my knee but not my ankle."
The Belgian started the year by winning the Australian Open for her fourth Grand Slam title, but has struggled since she overexerted her right shoulder and wrist during a busy spring schedule.
The 82nd-ranked Oprandi was stunned at her victory on the Rosmalen grass court.
"There is no bigger thing in tennis," she said. "It was a miracle."
Oprandi played with her right knee and arm heavily taped. She repeatedly surprised Clijsters with heavily sliced backhand drop shot winners, many of them off Clijsters' serve.
"I think maybe she was not 100 percent, and I tried drop shots," Oprandi said of her winning tactic. "It came out good for me."
It was Clijsters' second straight second-round exit, after losing to Arantxa Rus in the French Open.
Oprandi twice played drop shot winners off Clijsters' serve as she broke in the fifth game of the first set. However, Clijsters rallied to break Oprandi back in the next game.
But the Italian retained her composure to hit two aces and another drop shot winner to win the tiebreaker.
Oprandi again broke Clijsters in the second game of the second set and held her serve to record her biggest career win.
Clijsters' defeat means the top seeds in the men's and women's draw have both been knocked out. Michel Berrer of Germany beat Spaniard Nicolas Almagro 3-6, 7-6 (5), 6-4 in a first-round match on Monday.