Streelman had seven birdies at Innisbrook's Copperhead course in Palm Harbor, Florida, where he's chasing a third PGA Tour title but his first since the 2014 Travelers Championship.
Roy, ranked 348th in the world, put himself in the hunt for a first tour title with a 65 that featured eight birdies and two bogeys.
China's Carl Yuan, Canadian Adam Svensson and American Peter Malnati were tied for third on 66.
Streelman kickstarted his round with a nine-foot birdie at the 11th. He rolled in a five-footer at the 14th and a 14-footer at 17 and two pairs of back-to-back birdies coming in - at the first and second and the fourth and fifth holes.
"One of those rounds that just kind of got off to the right start," said Streelman, who added he got "up and down a few times when I needed to" to keep the round going.
"I knew today was going to be the best weather day and then it's going to be hold on to your shorts here for the next few days," Streelman said. "So trying to just keep a good attitude and have some fun."
Hot putter
Roy made the most of a hot putter in a round that started at the 10th and included birdie putts of 20 feet at 17 and 18.
He nabbed an 18-foot birdie from the fringe at the third and chipped in at the sixth to reach six-under for the first time.
After a bogey at the seventh, he rolled in a 15-foot birdie putt at the par-three eighth to finish the day in solo second.
"I definitely rolled in a couple long ones, but I think the key was I made the shorter ones that you kind of have to make around here," he said. "Once I saw one of the longer ones go in I kind of calmed down a little bit. I had great pace all day on the greens, so never really had any super stressed-out par saves."
Of the birdie putts he made, Roy said the one on 18 was "a huge bonus."
"I kind of dripped one in there from 20 feet or so with a huge breaker. That was nice to see in," he said.
Yuan, celebrating his 27th birthday, played alongside Streelman and said they fed off each other.
"We both played some great golf and it was definitely good to have a group like that," said Yuan, whose round was highlighted by a 15-foot eagle at the 14th.
He said a "perfect four-iron" off the tee left him a "pretty big, breaking putt.
"But I practiced that putt in the practice round, so I kind of know what it was going to do," Yuan said.
Svensson, who won his only tour title at the 2022 RSM Classic, is looking for a bounce-back week after missing the cut each of the past three weeks.
"I have a pretty good, positive attitude," Svensson said. "I feel like if you're playing bad, you just got to play good some time, so just keep my head down and keep going."
