Nadal's fidgeting, shorts-tugging preparation before serves is a famous quirk of the Spaniard's game but it fell foul of watchful Greek umpire Evanthia Asderaki, and at exactly the wrong moment.
With the third set locked at 4-4 and deuce, the world number one's breach of the 20-second time limit saw him forfeit his first serve and throw away the ball in disgust in a rare loss of composure at Rod Laver Arena.
Nishikori duly won the next two points to capture the break and leave the Spaniard muttering as he trudged back to his chair, throwing a withering look at Asderaki.
Clearly fired up, Nadal struck back the next game to put the match back on serve and punished the profligate Nishikori in the tiebreak to set up a quarter-final against red-hot 22-year-old Grigor Dimitrov.
Nadal later complained that he had been given no warning before being slapped with the second time violation.
"The rules say you can do it, but in my opinion that goes against the show, that goes against the fans," the top seed told reporters after closing out an attritional match in three hours and 16 minutes.
"The rules are there, sometimes I accept the rules, sometimes I am wrong.
"I will try to go quicker for the future but it's important to have people on the chair who understand the game."
The brush with the chair umpire capped an eventful afternoon for the 13-times grand slam champion, who suffered the indignity of being broken four times having not surrendered serve in his previous three matches.
After the second of the breaks arrived in the second set, Nadal took a tumble on the baseline but instinctively rolled to protect the knees that have troubled him throughout his career.
The lace on one of his sneakers did not escape damage, however, forcing Nadal to scurry off the court for five minutes to grab a new a pair from his locker.
Nadal also struggled with a monstrous blister on the palm of his racquet hand, adding to his travails during a fiercely contested match.
Nadal had come hurtling into the match with a brutal demolition of Gael Monfils, but encountered a far different beast in 16th seeded Nishikori.
Dictating the play for large stretches of the match, the clean-hitting Japanese demanded his opponent's stoutest defence as he moved the Mallorcan around the court and struck 36 of the crispest winners.
"Always it's tough against him in every surface. He's a very complete player," Nadal said. "He's very quick. He's able to do the most difficult thing in tennis, that's taking the ball early and changing the directions.
"Kei is a potential top-10 player. So that's the real thing. It's a great victory for me."
Beaten by a string of deft Nishikori drop-shots, Nadal returned fire with a series of sizzling forehands down the line to capture the second set before motoring to a 4-1 lead in the third.
A capitulation appeared likely but Nishikori roared back to win five straight games in a scrambling revival that would have impressed his watching coach Michael Chang, a famous hustler.
But as has happened so many times before, Nadal found another gear when required to bring the match back on serve and the weary Japanese surrendered in the tiebreak in a hail of unforced errors.
Dimitrov reached his first Grand Slam quarter-final courtesy of a 6-3 3-6 6-2 6-4 win against Roberto Bautista Agut.
The Bulgarian sealed his progression in two hours and 24 minutes against Bautista Agut, who beat Juan Martin del Potro in round two.
"I'm really happy to be in the quarter-finals," said Dimitrov.
"I've been working really hard in the past year, especially in the off-season. So it's nice to see this result."