Three-time champion Djokovic took the first set 6-3 on Centre Court but began to lose his cool in the second as Nishikori battled back.
The Japan star secured an early break and then held from being 0-40 down in a lengthy game that infuriated Djokovic.
As he returned to his chair, Djokovic bounced his racquet off the baseline and received a Code of Conduct warning from umpire Carlos Ramos.
Djokovic did not take the violation particularly well, however, and confronted Ramos.
He said: So youre saying basically that if I hit a ball and then ruin the court I get a warning?
You think I ruined the court for just throwing my racket [down]? Do you think that or not? Be honest.
He then listened to the umpires response before shaking his head and muttering: Come on.
Former champion John McEnroe was sympathetic to Djokovic, saying on commentary for the BBC: You cant give him a warning for that, come on!
After Nishikori took the second set 6-3 to level the match, McEnroe added: This is big for Djokovic. Hes been brittle with the comeback.
How badly does he want to pay a Nadal or Federer? Does he believe he can play those two?