Villarrial is considering surgery to repair an overextended tendon in his right ankle, while Neufeld is out with an injured left foot that may also require surgery, the team announced. Both were hurt in Buffalo's 17-16 loss at Indianapolis last weekend.
The Bills (3-6) will consider promoting tight end Brad Cieslak off the practice squad but likely won't make any moves until Friday or Saturday.
"We do have some decisions we need to make," coach Dick Jauron said following Thursday's practice. "They don't have to be immediate. We may do something quickly, we may not."
Villarrial aggravated an injury that forced him to miss a game earlier this season. An 11-year veteran and the team's most experienced lineman, he's a three-year starter since joining Buffalo as a free agent. He spent his first eight seasons with Chicago.
Second-year player Duke Preston replaced Villarrial against Indianapolis and will make his second start at right guard this Sunday at Houston (3-6).
"He played pretty well in the game on Sunday," Jauron said. "We need to see him and we need to evaluate him."
It will mark the latest change to an already patchwork line that underwent a major shuffle during the Bills' bye three weeks ago. That's when Jason Peters was switched to left tackle, replacing Mike Gandy, who was moved to left guard. Seventh-round draft pick Terrance Pennington took over at right tackle, replacing Peters.
Neufeld is a six-year veteran who has been mostly used on special teams in his three-plus years with the Bills. His injury leaves Buffalo with two tight ends: starter Robert Royal and reserve Kevin Everett.
The Bills' list of players on IR keeps growing.
Meanwhile, reserve rookie linebacker Keith Ellison did not practice Thursday and is questionable for Sunday's game with a knee injury.