Quarterback Philip Rivers tried to push the ball down the field to Travis Benjamin in overtime but instead threw an interception to cornerback A.J. Bouye, who returned the ball to the Chargers' 1-yard line.
Rivers made a tackle on the play, and Jacksonville was penalized for taunting afterward, pushing the ball back to the 17.
The Chargers led for most of the game but dropped to 3-6 overall, and their playoff hopes appear to be on life support.
"We've lost some close games this year, but that's the first time that we've lost one like that," Chargers coach Anthony Lynn said. "To me, that was inexcusable. We had opportunities to win that game on the road, and we didn't finish plays. We had a couple of guys do some dumb things."
The latest loss continued a troubling trend of the Chargers faltering late in games. Since the start of 2015, the Chargers have lost 11 games that have been decided by three points or fewer -- most in the NFL, according to ESPN Stats ∓ Information.
The Chargers are 8-17 since the start of last season. Of those 17 losses, 14 have been within one possession -- also the most in the NFL in that span.
Entering Sunday, the Chargers had won six straight against the Jaguars and appeared to have this one put away in regulation. The Chargers' offense had the ball twice with the lead and with less than two minutes left, but couldn't put the game away.
"We know how big this one was," Rivers said. "This was a huge game, and really just felt like we were in such control the whole way. I know the score wasn't way out of reach, but we felt like we were in such control, and we were really all the way down to it.
"And give them credit, they found a way to win the game. These are the games we talk about winning, the close games. And we found another way [to lose] today -- crazy."
The Chargers had possession with 1:51 left on a first-and-15 from their 37. However, undrafted rookie Austin Ekeler fumbled after Malik Jackson hit his helmet on the football. Tashaun Gipson recovered, but the Chargers managed to keep the Jaguars from scoring when Tre Boston corralled his second interception of the game.
Los Angeles could not get another first down and punted. And Jaguars quarterback Blake Bortles managed to drive his team 36 yards on six plays, putting Jacksonville in position for Lambo's 35-yard field goal to force overtime.
Despite the fumble, Ekeler finished with 119 total yards from scrimmage and two touchdowns. He totaled five receptions for 77 yards -- including touchdown catches of 28 and 22 yards -- and added 42 rushing yards.
According to ESPN Stats ∓ Information, Ekeler became the first running back in the NFL with two touchdown catches of 20 or more yards in a game since Week 15 of the 2013 season when Jamaal Charles finished with three touchdown catches of 20-plus yards against the Oakland Raiders while still with the Kansas City Chiefs.
Ekeler's four touchdowns on the year tie him for second on the Chargers with Benjamin.