Webb, who drew to within one of Gustafson with birdies on the last two holes at Yarra Yarra Golf Club, posted a bogey-free 68 to finish at 11-under.
Gustafson had a two-shot lead at 11-under after she negated a birdie at the 15th with a bogey at the 16th. She rolled in a 40-foot birdie putt at 17 to return to 12-under, but Webb kept pace with a birdie at the same hole. Webb had a chance to even the score with 35-foot eagle attempt at the par-5 18th, but her putt missed the cup and she tapped in for a final birdie.
Although Gustafson made it interesting when she knocked her 12- footer for birdie four feet by the cup, she ran in the par-saving putt to avoid a playoff and capture her second title of 2001.
'It was tough out there battling against Karrie and I'm so happy to have come out in front again,' said Gustafson, who recorded a three-shot victory over the top-ranked Webb at the LPGA Tour's Memorial of Naples tournament in January.
'I certainly made it a little harder for myself at the end there. I was very nervous,' admitted the 27-year-old Swede. 'It was marginal whether I was in control for most of the day. Every time I go up against Karrie it's nerve-wracking because she is such a great player.'
After starting the day with a comfortable four-shot lead over Webb, Gustafson reeled off three straight birdies at the opening holes to extend her advantage to seven strokes at 14-under par. But Gustafson bogeyed the 5th and 6th holes to slip to 12-under, and Webb was able to narrow the margin to three shots with birdies at the 7th and 8th.
Gustafson's lead was trimmed to two when she bogeyed the 10th, but that was as close as Webb would get until the final hole. Webb had a chance to pull within a single stroke early on the back nine, but failed to convert on a short birdie putt at the 11th.
She later matched poor tee shots with Gustafson at the par-5 16th, a hole Gustafson went on to bogey, thus missing an opportunity to gain ground with a birdie or better.
'I can't be too disappointed,' said the 26-year-old Webb, who captured her fourth consecutive Australian Ladies Masters title a week ago. 'I shot a 68 and the ladies in the score hut said that was the lowest of the day. Sophie struggled a little bit in the middle of the round, but she played really well the last few holes. My hat is off to her. I did not do enough to win and she did.'
Gustafson's win in the early-season event in Florida was her first of the year and her third on the LPGA Tour. She broke through in the U.S. with a victory at last year's Chick-fil-A Charity Championship, then went on to win the Women's British Open at Royal Birkdale.
Sunday's victory moved Gustafson to the top of the Evian Ladies European Tour's Order of Merit. She is defending her title as Europe's top money earner from the 2000 season, as she was also the winner of last year's Irish and Italian Opens.
Jane Crafter, the 1997 Australian Open champ, struggled to a 2-over 74 Sunday but birdied the last hole to finish alone in third at 5-under 283. Fellow Aussie Corinne Dibnah turned in a 70 for fourth place at minus four.
Australia's Alison Munt, the leader after each of the first two rounds, rebounded from a third-round 78 by shooting an even-par 72. She tied for fifth place at 2-under with England's Alison Nicholas and Elisabeth Esterl of Germany.
England's Laura Davies, who also shot a 6-over 78 on Saturday, carded a 71 on Sunday that consisted of five bogeys, two birdies and a pair of eagles. She finished tied for 16th at 2-over par.