Harlan took a 21-9 advantage at the 7:45 mark of the third period when Chassidy Lawson hit two free throws to open the scoring in the second half.
But Barbourville cut the lead to one by the end of the third quarter, setting the stage for a down-to-the-wire finish.
Both teams exchanged baskets before a Courtney Bell jumper gave Harlan a 32-29 lead with 2:18 to play.
With 1:53 remaining, Kaitlyn Doan hit two free throws. Callie Mills' short jumper put the Lady Tigers ahead 33-32 with 1:18 left .
Sallie Broughton missed the front end of a bonus with 42.5 seconds to play. Kendra Latham missed a shot, and Broughton was fouled with 17.1 seconds remaining. The senior guard hit one of two as Barbourville led by two.
Brittany Jones missed a 3-point attempt with seven seconds left and Callie Mills was fouled with 5.3 seconds remaining. She hit one of two free throws, and Latham's 3-pointer at the buzzer fell short.
Doan, a senior guard, powered Barbourville (2-1) with 14 points. Callie Mills scored 12 points and grabbed seven rebounds.
"It took a good halftime speech from me," said first-year Barbourville coach Dennis Messer. "It's the first time this year I've got fired up on them, and they really responded.
"I told them we were down 10 points and had only scored nine points. We weren't looking to score. We might have been intimidated by Latham inside, and I kind of challenged our team to win the third quarter and the fourth quarter and pull it out."
Latham carried the load for the Lady Dragons, scoring 12 points, pulling down 16 rebounds and handing out four assists.
"I saw in today's (Harlan Daily Enterprise) that Harlan said their win over Cumberland was a statement game, and I kind of put our kids to the challenge," Messer said. "This is something we can built on throughout the year."
"My hat goes off to Barbourville and their coaching staff and players. They came out and wanted this game more than we did," said Harlan coach Chuck Mitchell.
"Not taking nothing away from them. I just thought we played a lackluster game on the defensive end and didn't play a good offense end either."
The Lady Dragons missed 11 of 14 shots in the first eight minutes but still led 7-6.
Harlan opened the second quarter by scoring seven unanswered points, including baskets by Latham and Charlene McMillen and a trey by Alexandria Allison.
Latham nailed a 3-pointer and Bell added a jumper inside as the Lady Dragons led 19-9 at the break.
"We've just got to get the ball inside to Latham more," said Mitchell. "Until we start hitting the outside jumpers it's not going to open it up inside."
Callie Mills scored seven points in the third quarter as Barbourville began to fight back.
Harlan (2-3) returns to action Tuesday against Middlesboro in the first game of a girls/boys doubleheader. Barbourville (2-1) plays host to Pineville on Monday.
Dana Abner scored 13 points and Ashley Smith added 11 as Barbourville downed the Lady Dragons 36-28 in the junior varsity game.
McMillen and Allison each led Harlan (1-3) with 10 points. Belinda Patton tallied six points while Misty Belcher scored two.
Caroline Huff 0-5 0-0 0, Brittany Jones 2-13 0-2 5, Chassidy Lawson 0-4 2-2 2, Courtney Bell 4-10 0-0 8, Kendra Latham 5-9 1-2 12, Alexandria Allison 1-2 0-0 3, Charlene McMillen 1-1 0-2 2. Totals: 13-44 3-8 32.
Sallie Broughton 0-2 3-5 3, Kaitlyn Doan 4-9 4-8 14, Callie Mills 4-12 4-8 12, Shaunda Mills 2-9 0-0 4, Whitney Mills 1-5 0-1 2, Ashley Smith 0-0 0-0 0. Totals: 11-37 11-22 35.
Harlan 7 12 9 4 32
Barbourville 6 3 18 8 35
3-point goals: Harlan 3-17 (Allison 1-1, Latham 1-2, Jones 1-8, Huff 0-3, Lawson 0-3), Barbourville 2-7 (Doan 2-4, C. Mills 0-3). Rebounds: Harlan 31 (Latham 16), Barbourville 26 (C. Mills 7). Assists: Harlan 9 (Latham 4), Barbourville 4 (Broughton 3). Turnovers: Harlan 13, Barbourville 14. Fouled out: Harlan (Huff).