Isner, a two-time champion in North Carolina, suffered an ankle injury in his third-round win over Mikhail Kukushkin.
The big-serving American pulled out of the ATP 250 event before his scheduled last-eight clash against Czech seventh seed Lukas Rosol.
It is particularly worrying for Isner ahead of the US Open, which starts on Monday.
"I am extremely disappointed to have to withdraw from the Winston-Salem Open, one of my favourite stops of the year," Isner said.
"It is extra disappointing for me given that it is my hometown tournament.
"I would have loved to continue to chase my third title here in Winston-Salem in front of these great fans, my friends and family."
Rosol will face Chinese Taipei's Lu Yen-Hsun in the semi-finals after the ninth seed eased past Italian 14th seed Andreas Seppi 6-4 6-4.
The other semi-final will be a meeting between two unseeded players.
Talented Pole Jerzy Janowicz had no problems beating Goffin 6-4 6-2, ending the Belgian qualifier's winning run at 25 matches.
He will face Sam Querrey after the American battled past Spanish fifth seed Guillermo Garcia-Lopez 6-7 (3-7) 6-2 6-4.