McIlroy took a one-shot advantage into the third round at a soggy Valhalla and held onto it thanks to a second successive 67 to lie 13 under par, but the identity of his nearest challenger came as something of a surprise.
Wiesberger was a total of 12 over for his last two appearances in the US PGA before this week and had only made one halfway cut in five previous majors, but carded a flawless 65 which was completed in stunning fashion.
The 28-year-old world number 70 holed from inside three feet on the 505-yard 16th, almost holed his second shot to the 17th and then left an eagle pitch just inches short on the 18th.
"It was a dream come true really, going out there with Phil (Mickelson), one of my heroes," said Wiesberger, who lost a play-off for the Lyoness Open in his native Vienna in June, the event he won in 2012 for his second European Tour title.
"I played beautifully today, I didn't miss a lot of shots, set up a few nice opportunities on the last few holes especially. I'm very proud of myself the way I played.
"It's a completely new situation for me, only my second cut in a major in six attempts, so I am quite a rookie in this particularly situation. I have driven it really nicely this week and if I can do it again it will settle down the nerves and I am going to have fun tomorrow."
American Rickie Fowler, who has finished in a tie for fifth, second and second in this year's majors, was a shot behind Wiesberger on 11 under with Mickelson another stroke back after both shot 67. Australian Jason Day was alongside Mickelson after a 69.
McIlroy, who won the Open Championship at Royal Liverpool and a first World Golf Championship event in the Bridgestone Invitational on Sunday, is aiming to become the first player since Padraig Harrington in 2008 to win back-to-back majors.
The 25-year-old won his first two by eight shots and was six ahead after 54 holes at Hoylake but was happy simply to still be in the lead here.
"The guys got pretty close to me at the Open and today and I was able to respond on the back nine," McIlroy said. "It's not the biggest lead I've ever had but I am still in control of this tournament and it's still a good position to be in.
"I just knew I needed to make a couple (of birdies) coming down the back nine to keep the lead or at least be tied. The two birdies on 15 and 16 were huge.
"It's where I want to be, it's the best place to be in a tournament. I wouldn't want to be anywhere else."
McIlroy's Ryder Cup team-mate Graeme McDowell felt the course was playing more like a regular PGA Tour event than a major championship, but the final pairing of McIlroy and Day began their rounds scrambling for dramatic pars.
Day hooked his tee shot on the second so badly that it cleared Floyd's Fork, the creek running down the left-hand side of the hole, and into deep grass on the far side.
Television commentator David Feherty initially looked to be searching for the ball in order for Day to work out where he could take a penalty drop, but the Australian then sent his caddie Colin Swatton to wade through the creek to assist.
When the ball was found in a good enough lie for Day to be able to play it, the 26-year-old then took his shoes and socks off as well and made the journey across to the other side. Instructing Swatton to "throw me a pitching wedge", Day duly hacked out of the rough, pitched onto the green and holed from 12 feet for a remarkable par.
Two holes later it was McIlroy's turn to escape with a par after pulling his drive into a hazard on the par-four fourth, which had been reduced to 292 yards to allow players to try to drive the green.
After taking a drop McIlroy pitched to 11 feet and holed the putt, although he was joined in the lead by Day who was left with a tap-in birdie after missing from close range for an eagle.
Both players then made birdie on the fifth but McIlroy reclaimed the lead when Day bogeyed the sixth after a wild drive and the world number one was two clear when he holed from five feet on the par-five seventh for birdie.
However, McIlroy duffed his chip from the edge of the eighth green and a repeat on the 12th meant he was briefly a shot behind Wiesberger when the world number 70 birdied the 16th and almost holed his approach to the 17th.
However, McIlroy responded in stunning style, holing from 20 feet on the 15th and then reducing the 505-yard 16th to a drive and a nine-iron which stopped two feet from the hole.
"Hmmm, drive and a 9 iron into 16 £wow," Luke Donald wrote on Twitter and after Wiesberger had birdied the last there was another massive drive from McIlroy on the same hole to set up a closing birdie from a greenside bunker for the outright lead.
Official World Ranking
1 Rory McIlroy (Nirl) 9.40pts ave, 2 Adam Scott (Aus) 9.23, 3 Sergio Garcia (Spa) 7.78, 4 Henrik Stenson (Swe) 7.66, 5 Justin Rose (Eng) 7.46, 6 Matt Kuchar (USA) 6.77, 7 Bubba Watson (USA) 6.66, 8 Jim Furyk (USA) 6.47, 9 Jason Day (Aus) 6.19, 10 Tiger Woods (USA) 5.90, 15. Graeme McDowell (Nirl) 4.90; 58 Shane Lowry (Irl) 2.25
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