The bizarre incident happened between games in the first set, with the Spanish great leading 4-3 against Britain's Jack Draper at Rod Laver Arena.
"I need the racket back," the 36-year-old defending champion, looking bewildered, told the chair umpire.
It was not immediately clear what happened, but it seems that Nadal asked for his racket to be restrung, only for the ball boy to take the wrong one.
"The ball boy took my racket!" a smiling Nadal shouted to Draper, explaining what the hold-up was.
It did not overly affect the 22-time Grand Slam champion as he went on to win the match, despite a second-set wobble, 7-5, 2-6, 6-4, 6-1.
"Not distracting, just a funny situation," Nadal told Eurosport later.
"I said 'I need the stringer', but I needed the stringer to look at the other rackets.
"So he picked up my racket I was ready to play (with). But okay, he took it and no problem at all."
Nadal next plays the American Mackenzie McDonald at Melbourne Park.
