The 46-year-old American, bidding to become the oldest winner of the championship in the modern era, compiled a two-under-par 69 to finish 10-under on 132.
Swede Henrik Stenson (65) was one shot off the pace while Dane Soren Kjeldsen (68) and American Keegan Bradley (68) shared third place on 135.
Conditions were perfectly acceptable on the front nine for the morning starters and Mickelson took advantage with early birdies at the fourth and seventh.
The veteran left-hander then delivered a pinpoint tee shot at Royal Troon's signature eighth hole, the par-three Postage Stamp, that landed several feet beyond the flag before spinning back to stop within inches of the cup.
As driving rain began to fall on the inward half, Mickelson mixed two bogeys with a solitary birdie.
"I really enjoy the challenge this weather and these elements provide," the 2013 British Open champion told reporters. "I thought it was a good round to back up the low round (63) yesterday.
"I made one or two bad swings that led to bogeys but for the most part kept the ball in play and played kind of stress-free golf.
"One of the things I've really worked on over the years is getting the ball on to the ground off the tee quickly so the ground is affecting it more so than the air," added Mickelson.
The weather deteriorated in the afternoon and none of the players in the second half of the draw were able to mount a charge.
'Great result'
Stenson's six-under 65 was the best round of the day and he was delighted to reel in Mickelson after the American had begun the day five shots ahead of the Swede.
"I was hoping to gain a little and the way it turned out I gained quite a lot," said world number six Stenson after firing seven birdies.
"I'm happy with the way I played of course. It's not easy out there. It's a great result."
The so-called 'Big Four' of world golf were again left trailing in the wake of the leaders.
World number two Dustin Johnson (69) and fourth-ranked Rory McIlroy (71) were eight behind Mickelson. Number one Jason Day (70) was 11 adrift while third-ranked Jordan Spieth (75) was four-over.
There was a sombre mood at the start of the day with the French tricolour flying at half-mast by the 18th green as the Open paid tribute to the victims of the Bastille Day attack in Nice on Thursday evening.
French golfer Clement Sordet, who teed off in the first three-ball, had the words 'pray for Nice' emblazoned on his cap which also carried a black ribbon.
"I'm really proud to be French and I think we all need to support each other. I just say my prayers," added Sordet.
Stenson, former world number one Lee Westwood and four-time major winner Ernie Els were among several other players wearing black ribbons on their headgear.
