Matthew Tkachuk scored, and Brandon Montour had two assists for the Panthers (40-31-7), who have won four in a row and moved into a tie with the New York Islanders for the first wild card into the Stanley Cup Playoffs from the Eastern Conference, one point ahead of the Pittsburgh Penguins, who lost 5-1 at the New Jersey Devils on Tuesday.
Devon Levi made 34 saves in his second NHL start, and Dylan Cozens scored for the Sabres (37-32-7), whose five-game point streak (4-0-1) ended.
Buffalo trails the Panthers and Islanders by six points in the wild card race.
"Someone just mentioned to me we had nine guys in the lineup under 23 compared to one under 23 [for the Panthers]," Sabres coach Don Granato said. "So, there's a difference there. And for our guys, they just have to go through it. And I think they're better for it.
"I know they're ultracompetitive. You can't hang your head, you got to move right on. That was disappointing tonight, but I still fully believe we have a chance and I think our guys do. And there's more meaningful games ahead, starting with Thursday [at the Detroit Red Wings]. We move on to that quick but take some experience from this one, without question."
Tkachuk gave Florida a 2-1 lead at 59 seconds of the third period when he deflected Montour's shot from the point for his 39th goal of the season.
"I think it is just easier playing with the lead, so getting that second goal, giving ourselves a lead was huge for us," Tkachuk said. "It was pretty even and just back-and-forth. Both goalies were great. We capitalized on strong, hard plays in the third and Lyon made some big saves."