Padraig Harrington was trailing leader by just one stroke after the third round of the Nissan Open in California.
Mickelson shot a two-under-par 69 to go to 13-under par to lead Harrington with Rich Beem two strokes behind and Robert Allenby and Charles Howell III three strokes adrift.
Harrington and Mickelson began the day tied for the lead at 11-under par, but two quick birdies by Mickelson gave him the advantage.
He nearly reached the par-five first hole in two shots, his second settling on the fringe 97 feet away and then two-putting. He added another birdie on the next hole when he made a five-foot putt.
On the back nine, Harrington added a birdie on the 11th hole and then made pars the rest of the nine to settle for a one-under 70.
Mickelson made bogeys on three of five holes.
Beem had the best round of the day, a six-under 65 to get to third place.
The 36-year-old American has not won since he captured the 2002 PGA Championship.
David Howell struggled in the third round, shooting a two-over par and dropping to a tie for 19th.