Molinari canned a 44ft birdie putt on the last hole at Bay Hill to card a closing 64 and post 12 under, which ultimately gave him a two-shot victory over Fitzpatrick.
Molinari hit 12 of 14 fairways and 14 of 18 greens in regulation, and was equally impressive on the greens, holing every putt he faced within 10ft.
At the 16th he reached the green in two, leading the way for a two-putt birdie at the par-5 hole.
Fitzpatrick had taken a one-shot lead over Rory McIlroy into the final round. After back-to-back 70s in the opening two rounds he posted five birdies and dropped no shots in a 67 on Saturday to move to nine under par.
He birdied the fourth and parred the other eight holes on his way to the turn in the final round, but found himself overtaken by Molinari.
Fitzpatrick’s run of 10 consecutive pars was ended on the 15th, unfortunately with a dropped shot. He retrieved it at the next and closed with a one-under-par 71 to take second spot behind Molinari.
South Korea’s Sungjae Im, England’s Tommy Fleetwood and Spain’s Rafa Cabrera Bello tied for third, one back of Fitzpatrick.
South Africa’s Justin Harding is within sight of securing a Masters debut after claiming his first European Tour title in the Qatar Masters.
Harding birdied three of the last four holes in a closing 66 to finish 13 under par, two shots ahead of a nine-strong group which included compatriots George Coetzee, Christiaan Bezuidenhout and Erik Van Rooyen and England’s Oliver Wilson.
Wilson, who began the day with a one-shot lead, carded a closing 71 at Doha Golf Club.
Harding, who won twice on the Asian Tour and twice on the Sunshine Tour in 2018, is projected to move just outside the world’s top 50 when the rankings are updated today. The top 50 on April 1 receive an invite to the year’s first major at Augusta National.
Harding was three shots off the lead at the start of the day and although he birdied three of the first four holes, bogeys at the sixth and seventh looked to have derailed his title challenge.
However, the 33-year-old birdied the 10th, 12th, 15th, 17th and 18th in a back nine of 31 to post a clubhouse target that no-one was able to match.
England’s Meghan MacLaren claimed back-to-back titles in the Women’s New South Wales Open with a three-stroke victory at Queanbeyan Golf Club.
The 24-year-old, from Northamptonshire, went into day four tied at the top of the leaderboard with Lynn Carlsson before carding a closing 69 to finish 12 under par.