The tournament in Indian Wells was Djokovic's first since winning his seventh Australian Open title in January and the 31-year-old said he had already put the defeat behind him and turned his thoughts to next week's Miami Open. I don't know. I'm still in the tournament feeling great, playing good tennis.
He had won eight of nine prior meetings with Kohlschreiber, but he could find no answer for the German.
"It's a very special moment", Kohlschreiber said.
While it is being assumed it was just one of the bad days for Nole, there are debates that Nole was under pressure due to off-field controversy.
He came out with a crisp, confident style, breaking Djokovic in the seventh game and closing out the opening set with a service victor.
Djokovic and Fognini got the first break of the match when they broke Rojer and Tecau in the fifth game to take a 3-2 first-set lead.
Djokovic led that game 30-15, but capped a long rally with a forehand long then double faulted before pushing a forehand wide on break point.
He made no mistake on his second chance, however, firing a sideline-skimming forehand to seal the milestone win on his first match point.
Next up for Kohlschreiber is Gael Monfils, who owns a 13-2 record against the German.
The German punched the air in celebration after a video review confirmed that his forehand victor caught the line on match point to seal the upset victory in Indian Wells. It's part of our world, and we just have to bounce back very quickly. "He can play well on any surface and I think he should get more recognition because he's done a lot for the sport as a whole".
"Things to work on, without a doubt", Djokovic said. "So it's a positive victory for me".
The Serb, who now holds three of the four Grand Slam titles, was beaten 6-4 6-4 by the German world number 39.
The bottom half of the men's draw still includes No. 2 seed Rafael Nadal and fourth-seeded Roger Federer, both of whom breezed to victory.
"Don't remind me of the stats", Wawrinka quipped after pulling off a marathon victory over Hungarian Marton Fucsovics. Djokovic returned about an hour after his loss for their match.
Krajinovic will be a big underdog when he faces Spain's world number two Rafa Nadal, who crushed Argentine Diego Schwartzman 6-3 6-1 on the showcase court to reach the last 16.
Muguruza next faces 18-year-old Canadian Bianca Andreescu, who surprised China's 18th-seeded Wang Qiang 7-5, 6-2.