Gustafson fired a final-round 2-under-par 70 to hold off any challenge from her Sunday playing companion, who shot 69. At 16-under, the 27-year-old Swede tied the tournament record, originally set by Meg Mallon in 1999.
Gustafson entered the final round at The Club at The Strand with a commanding four-shot lead over Webb. She extended her advantage to five by dropping a 10-foot birdie putt at the first, and maintained a comfortable margin throughout the day.
Webb tried to make a late rally, birdying the 11th 12th and 14th holes, but bogeys at the 13th and 16th levied the pressure. Webb finished her round by recording her sixth birdie of the day at the par-3 17th that enabled the Aussie to finish in solo second place at 13-under-par.
It's Karrie's 20th career runner-up finish on the LPGA - compared to 23 victories.
'I'm very happy to finish second and am glad I ran out of holes out there today because I did not hit the ball the way I like,' said Webb, who earned $93,093. 'I had some chances out there today and just didn't take advantage of my opportunities.
'Sophie's game was right on this week and she did well and deserves to win.'
Despite being paired in the final group with the world's top female player, Gustafson bettered Webb on the front nine by one shot, and then kept her at arm's length on the back with birdies at the 12th and 13th holes.
For Gustafson, who collected $150,000, the win is her third in just her third full season on Tour. Last year, Gustafson won the Chick-fil-A Charity Championship and the Weetabix Women's British Open.
Dottie Pepper (67) finished in third place at 12-under, while Marisa Baena (66) tied her career-best performance on Tour with a fourth-place finish at 11-under.
Juli Inkster (70), Laura Davies (66), Michele Redman (67), Jean Bartholomew (66) and Vicki Goetze-Ackerman (70) tied for fifth place at minus-10.
Click here for Full-Field scores from the Subaru Memorial of Naples!