Europe captain Luke Donald, meanwhile, made a novice's error after hitting into the thick rough at the Marco Simone club outside Rome and then hitting the wrong ball, earning himself a two-stroke penalty.
McIlroy (66) produced an eagle for the second consecutive day and Fitzpatrick (69) had five straight birdies in the second round Friday as they stood one and two, respectively, near the midpoint of the Italian Open. The round was suspended because of darkness for the second straight day.
McIlroy - playing in Italy for the first time as a pro specifically to gain familiarity with the Ryder Cup course - was at 9-under 133 overall with Fitzpatrick one stroke behind.
After McIlroy took a share of the overnight clubhouse lead with an opening 67 before play was cut short Thursday, Fitzpatrick had a strong finish early Friday to take the lead after the first round with a 65.
Later in the day, it was McIlroy who surged ahead as he sank an eagle putt on the par-5 12th and celebrated with a slight wave - unlike his arms-above-the-head reaction when he holed out an approach shot for an eagle on the par-4 third a day earlier.
Fitzpatrick, who claimed his first major title at The Country Club in Brookline, Massachusetts, in June, also tied for fifth at this year's PGA Championship. At 28, he's a veteran of the European Tour and this is the seventh time he's playing the Italian Open, having finished second in 2019.
"I love Italy," Fitzpatrick said, adding that he's a big football fan. "I came in December to watch Roma."
Donald struggled to a triple bogey on the driveable par-4 16th when he hit his tee shot into the rough and then played the wrong ball.
"A first for me," Donald wrote on Twitter. "Poor drive in deep rough, spotter goes straight to ball, play the shot and then realise I've played the wrong ball. Same red Titleist 4, just not my markings on the ball."
Donald, however, recovered by making birdies on five of the next eight holes to finish in contention at 5 under.
Afterward, Donald suggested that the rough will be "pretty high" for the 2023 Ryder Cup - when Marco Simone will become the third venue in continental Europe to host golf's biggest team event after Valderrama in Spain (1997) and Le Golf National in France (2018).
"We want to put a premium on hitting the fairways," Donald said. "The rough here, it's a different type of grass. It's kind of a thick blade, so it doesn't need to be too, too heavy to make it tough. ... We obviously want to play similar conditions to what we played in this year."
Defending champion Nicolai Hojgaard (70) also remained in contention at 5 under, with 10th-ranked Viktor Hovland (68) a stroke further back. Tom Lewis was third at 7 under and Victor Perez stood fourth at 6 under.
Local player Filippo Celli continued to impress in his pro debut after a sterling amateur career, scoring an eagle with a wedge shot on No. 12 to finish at 1 under and make the cut.
Julien Quesne made a hole-in-one on the 187-yard fourth.