Woods drained a breaking 15-foot birdie putt on the 18th green to outlast Mickelson, who earlier in the round, saved par beautifully on the final hole when his third shot landed five feet from the cup.
Ernie Els held on to third, followed by Lee Westwood and Davis Love III, who did not play at Bay Hill citing fatigue.
Colin Montgomerie stayed sixth this week while David Duval took seventh. Duval withdrew from the Bay Hill Invitational due to a wrist injury.
Vijay Singh, who finished in a tie for fourth at Bay Hill, remained eighth followed by Hal Sutton, who will defending his Players Championship title this week in Florida. Tom Lehman rounded out the top-10.
Darren Clarke played horribly at Bay Hill, when he missed the cut with a 14-over 158 but held on to the 11th ranking from last week. Jesper Parnevik, Nick Price, Jim Furyk and Thomas Bjorn also stayed where they were last week, completing the top-15.
Sergio Garcia, who shared fourth in Florida on Sunday, jumped one place to 16th while Mike Weir dropped one to 17.
Ireland's Padraig Harrington also climbed one notch to 18 while Michael Campbell fell one to number-19. Stewart Cink came in 20th.
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