Glover and Nitties opened with matching six-under-par 65s while Mickelson launched his 2009 PGA Tour campaign with a double-bogey at the first before completing an ugly 76.
"Well, it really wasn't what I was hoping for," the American world number four told reporters after making his 18th consecutive start at the TPC Scottsdale. "I had three penalty shots, and that hurts.
"But it didn't feel that far off," added Mickelson, who won the tournament in 1996 and 2005 and was edged out by compatriot JB Holmes in a playoff for last year's title.
"I felt like I hit the ball pretty solid. I know the score is ridiculous but it doesn't feel bad. I feel like I can come back out tomorrow and shoot a good round and play the weekend.
"I probably have to shoot about five under but that doesn't feel out of the question."
While Mickelson piled up two double-bogeys, three bogeys and two birdies on Scottsdale's Stadium Course, Glover and Nitties played near-flawless golf to charge into the lead.
Glover, whose only PGA Tour victory came at the 2005 Disney Classic, mixed seven birdies with a lone bogey at the 17th to join Nitties at six under.
High Stuff
"It was pretty solid," the 29-year-old said. "I hit a lot of fairways, a lot of first cuts (of rough) actually and only had a couple in the high stuff.
"But I played well. I made a couple of nice birdie putts and a couple of key par putts. I also played the par-fives well and that usually equals a good score. I was pretty pleased."
Rookie Nitties, who competed in the PGA Tour's qualifying tournament each year since 2005 before finally breaking through in December, was also delighted after setting the early pace.
"I hit it pretty solidly and I putted well today," the 26-year-old said. "I didn't really get into too much trouble all day and, if I did, I'd sort of make the smart decision. It worked together well."
Nitties, who lists socializing and girls among his chief interests, feels completely at home at the Phoenix Open, which is known for delivering the biggest party on the circuit.
"This was one of the events I was looking forward to the most," he said. "If you knew me, it's tough for me to concentrate out here.
"But on the flipside, I'm on the PGA Tour and I'm not going to take it for granted. I know when to put my partying on hold and I know when to put in hard work."
David Berganio junior and Charley Hoffman opened with 66s to end the day a stroke in front of fellow Americans Briny Baird, Kevin Na and Matt Kuchar.
Defending champion JB Holmes, who rattled up six birdies in seven holes to reach the turn in five under, lost momentum on his back nine to card a 70.
