The American signed for a sizzling nine under 63 around Bay Hill, enough for a three-stroke lead over the red-hot Collin Morikawa and Sweden's Ludvig Aberg.
Berger had the benefit of racing around a benign Bay Hill in the morning before the wind picked up and made it a challenge for the afternoon field. He fired four birdies going out and added another five on the back nine, including a gain on the final hole.
"It has that US Open kind of feel to it," Berger said of Bay Hill.
"I think the course is just going to get tougher as the week goes on. The greens are going to get firmer. So yeah, just hit it in the fairway and try to make as many putts as you can."
Morikawa, who is coming off winning at Pebble Beach and a T7 at the Genesis Invitational two weeks ago, enjoyed a fast finish of eagle-birdie-birdie to fly up the leaderboard
Meanwhile, Justin Thomas endured a rude welcome back from five months away recovering from back surgery. His seven over 79 included a double bogey on the par four 11th when he missed the fairway and missed a three-foot putt, and another double on the 16th when a wedge from 81 yards splashed into the creek.
"It was kind of hard to say it was good to be back out," said the two-time major winner.
"Yeah, not obviously how I expected it to go. But the rust aspect kind of unfortunately was a little bit of what I anticipated."
Matt Fitzpatrick and his younger brother Alex held their nerve down the stretch to secure a landmark win at the Zurich Classic of New Orleans, a triumph that also hands Alex a two-year PGA Tour exemption.
Star pairing Brooks Koepka and Shane Lowry fell short of the leaders at the Zurich Classic of New Orleans, missing the cut by a single shot as they exited before the weekend.
Bryson DeChambeau has reaffirmed his commitment to LIV Golf amid ongoing speculation about his future, saying he intends to remain with the league if it continues beyond this season.
Americans Alex Smalley and Hayden Springer lead the PGA Tour's only team event, the Zurich Classic, by one stroke.
