Hughes, who has a history of verbally admonishing game officials, was fined $23,152 Friday for abusive language toward an official. He also was docked $8,681 for unnecessary roughness for head-butting Giants rookie tackle Ereck Flowers.
Rams WR Stedman Bailey used the football as a pillow after catching a touchdown pass at Arizona. That cost him $8,681 for unsportsmanlike conduct.
Hughes lost his cool on two occasions against the Giants, when the Bills were flagged 17 times for 135 yards in their loss.
First he was penalized in the second quarter for head-butting Flowers following Rashad Jennings 14-yard run.
Then, the Bills lost their composure during a fourth-quarter drive. Linebacker Preston Brown was penalized for unnecessary roughness on the first play from scrimmage. Before the Giants snapped the ball again, Hughes was flagged for unsportsmanlike conduct.
Hughes has a track record for undisciplined play.
Last year, following a 24-17 loss at Denver, Hughes said he learned his lesson and vowed to keep his mouth shut after being penalized a third time for arguing with officials.
Upset with a pass interference penalty against cornerback Stephon Gilmore, Hughes voiced his complaints to an official and was flagged 15 yards to extend a drive that led Denver build a 14-3 lead.
"That's just a boneheaded play by me," Hughes said, afterward. "I've learned from my mistakes. You've got to walk away."
Hughes is a part of what the Bills expect to be a dominant defensive front, and was re-signed by the team to a five-year contract worth more than $45 million in March.
Penalties have become an issue for the Bills under new coach Rex Ryan.
They were also flagged 14 times for 140 yards in a 40-32 loss to New England on Sept. 20. The Bills lead the NFL with 47 penalties for 428 yards.
Ryan has made it a focus to reduce penalties this week. When a player commits a penalty in practice, they are forced to stand and watch as the rest of the team does 10 pushups.