A free-flowing Molinari sank 10 birdies in a 10-under 62 to lead Spain's Alejandro Canizares (64) by two shots.
Denmark's Soren Kjeldsen and Frenchman Raphael Jacquelin (both 65) are one shot further back on seven-under.
Donald, in his 52nd week as the world's top-ranked golfer, was seven-under through 13 but bogeyed two of his final five holes.
"Usually I'm pretty happy with 67, but I'm five back already," Donald told the European Tour website.
"But there were a lot of positives there after three weeks away from competition."
Donald, chasing a maiden Major title, is playing for the first time since last month's U.S. Open where he missed the cut.
Molinari said he hoped his dazzling round would raise the spirits of his brother Edoardo, who won the title at Loch Lomond two years ago but is recovering from wrist surgery this time.
"It would be nice to do well this week for him as well - to cheer him up a little bit," he said.
"I know it's not going to last forever," he added, referring to his hot form, "but I hope to keep this going a little bit longer - obviously next week, but the next month or so is really big for the Ryder Cup."
Four-times Major-winning American Phil Mickelson recorded a one-over 73, mixing four birdies with three bogeys and a double bogey.