The England captain condition's appeared to worsen while he was playing in Yorkshire's innings and 25-run County Championship First Division defeat away to Sussex at Arundel concluded Friday.
Vaughan, missing England's ongoing One-day series against Sri Lanka after being sidelined from the 1-1 drawn Test campaign earlier this season, has been ruled out of Yorkshire's C and G One-day Trophy match against Worcestershire at New Road on Sunday.
He spent much of the match at Arundel in the dressing room while Yorkshire were fielding. And when he came out to bat Friday he could only manage four runs off four balls before being lbw to Pakistan seamer Yasir Arafat.
"Michael's rehab programme seems to have suffered a setback and we will have to decide whether he needs a longer period of strengthening his knee or if some other course of action is required," Yorkshire physio Scott McAllister told Saturday's edition of the Northern Echo newspaper.
"A fourth operation on his knee is a possibility but at this stage nothing has been set in stone and the situation needs to be carefully assessed."
A fresh operation would likely sideline Vaughan, 31, from the remainder of the season with both Yorkshire and England, keeping him out of the Test series against Pakistan starting next month at Lord's.
And that would place a big question mark over whether Vaughan, who last year led England to their home Ashes series triumph, would be fit enough for the Ashes contest in Australia starting in November.
Vaughan has not played for England since the tour of Pakistan before Christmas. He only returned to cricket with Yorkshire a month ago and has repeatedly used ice treatment on his knee.
England, 2-0 down in their One-day campaign against Sri Lanka, had to avoid defeat at the Riverside on Saturday to keep the five-match series alive.