Nicholas Thompson (66) and Freddie Jacobson (67) tied for third at 8 under.
David Toms, in the final group, led at 9 under when he made his turn. But he had three bogeys over the next five holes and finished with a 70.
Toms ended up 7 under with Brendon Todd (68), who last week got his first PGA Tour victory at the Nelson. Jimmy Walker, No. 1 in the FedEx Cup standings, shot a 70 was tied for 10th at 6 under.
About 30 minutes after Dufner made his long birdie at No. 18 to get to 9 under, Scott made a closing par after just missing a 31-foot birdie chance that would have wrapped up the victory. It was just less than an hour later when the two went back to No. 18 to start the playoff.
Scott finished about the same time Toms was coming off bogey at the par-3 13th that dropped him two strokes back.
Scott was already 8 under after making a 6-footer at the 406-yard No. 6 hole, his fourth birdie in the round and already with a bogey at the par-4 third when he three-putted from 24 feet. His only other three-putt at Colonial came from 17 feet at the ninth hole, dropping him to 6 under.
"I didn't want to let this one slip," Scott said. "As always, a bit of luck involved. And to come out on top is a really satisfying feeling, and hopefully keep No. 1 for at least another week."
Dufner, who was also second at Colonial two years ago, gave up a stroke when he missed a par putt from less than 3 feet at the straight 615-yard 11th hole. When his 12-foot birdie chance at the 440-yard 12th hole slid a half-foot past the cup, Dufner stood momentarily and stared at the ball before tapping in his par.
Scott caught up with consecutive birdies on those same holes, pitching to 9 feet at No. 11 before his approach at No. 12 to 4 feet.
Dufner had another near miss at No. 15, where another lengthy birdie chance slid inches by the hole. Scott got to 9 under with a 39-foot birdie putt at the 453-yard 14th hole.