The winning putt wrapped up a closing 3-under 67 for an 11-under 269 total, one ahead of Dicky Pride.
Dufner got his first PGA Tour victory at New Orleans on April 29, then took a week off to get married. He also led alone by one stroke after the second and third rounds at the TPC at Four Seasons course.
Pride, whose only PGA Tour victory in a 20-year professional career came in 1994, was at 10 under with a par-saving 22-foot putt at No. 18 after hitting his drive into the water.
Moments later, Dufner made a putt that was only a few feet longer but on virtually the same line as the one Pride made to finish his round of 67.
J.J. Henry, who had an early hole-in-one, was in the lead at 11 under after consecutive birdies at Nos. 15 and 16, overcoming a bad tee shot on the first and a greenside bunker on the second.
But Henry, in the final group with Dufner, hit his tee shot at the par-3 17th over the green. The former TCU star lost the lead with a double bogey after a 4-foot putt slid just outside the cup.
After watching Henry's meltdown, Dufner made a tap-in par at No. 17 and then hit a big drive on No. 18 in the middle of the fairway. He hit his approach to the middle of the green, avoiding a playoff with the long putt and joining Hunter Mahan as the only two-time winners this season.
Pride had made a 13-foot birdie putt at No. 17.
Phil Mickelson, making his first Nelson start in five years, had four consecutive birdies on the front nine and went on to a round of 66 to finish four strokes back. He tied for seventh with Ken Duke, who also had a string of four birdies in a row in his own 66.
While Henry and Dufner were playing the 17th hole, Pride drove his tee shot at No. 18 left into the water. After his drop, Pride hit his approach to 22 feet and made that par-saving putt to remain at 10 under, letting out a scream when the ball dropped into the hole.
Pride, who went to the University of Alabama, then watched the final group play the 18th hole and waiting to see if he would be in a playoff. Even he had to applaud on the clinching putt by Dufner, who went to rival Auburn.
Matt Kuchar, the fifth-ranked player who won The Players Championship a week earlier, had 70 and finished at 276 in a tie for 15th. He was trying to become the first PGA Tour player since Tiger Woods in 2009 to win in consecutive weeks.
Henry's ace came at the 154-yard No. 5 hole, when he hit a pitching wedge. When the ball rolled back into the cup, Henry thrust both of his arms in the air, then had an emphatic uppercut and he celebrated with the gallery.
After Henry's drive at the 504-yard 15th was way right and under a tree, he hit his approach to the middle of the green and made a 32-foot birdie putt that led to another, though more subdued fistpump. His approach at the easier par-5 16th went in a greenside bunker, but he blasted inside 2 1/2 feet for a birdie to get to 11 under.
Then came the tee shot at No. 17 that carried over the green on the opposite side of the hole.
Dufner had consecutive bogeys at Nos. 2 and 3, but quickly responded with consecutive birdies on the following two holes.
After a bogey at No. 11 following a bad tee shot, Dufner got back to 10 under with his birdie at the par-5 16th, when he blasted out of a greenside bunker to 6 feet.
The last of Mickelson's four birdies in a row came when he holed a 30-footer at the 461-yard eighth hole. That got him at 6 under at about the same time the final group of Henry and Dufner were finishing at No. 1, both at 8 under.
Mickelson was 8 under after a birdie at the par-5 16th, but his 3 1/2-foot par putt at the following par 3 circled the cup and rolled back toward him for his first bogey of the day. He finished with another bogey at No. 18, though that could have been worse after his approach from the rough went into the water. After his drop, his 54-foot par-saving putt rolled over the cup, banged into the edge but didn't drop.
Vijay Singh, a 34-time PGA Tour winner whose last victory was in 2008, was within two strokes of the lead after his third-round 66. But he hit his first tee shot Sunday into the rough and his second into a greenside bunker on way to bogeys on his first three holes and a 71 for a ninth-place tie at 275.