Nicklaus, 79, won the tournament a record six times during his decorated playing tenure. Player, 83, won three times at Augusta National.
An excited gallery gathered around the golf legends. Augusta National Golf Club chairman Fred Ridley gave a speech at the first tee before introducing Nicklaus and Player.
Player -- who participated in a record 52 Masters tournaments -- took the first tee shot. The South African grooved the shot down the fairway, before celebrating with a leg kick.
Nicklaus joked and groaned as he bent over to place his ball on the tee, before ripping a shot down the fairway.
Player and Nicklaus also played together, along with Tom Watson, at Wednesday's Par-3 contest. Player tied for 18th by shooting a 1-over-par 28. Nicklaus' round was not scored.
"No aces this year and our trio didn't all finish in the top 9," Nicklaus tweeted. "But it was still 9 holes to remember in the Masters Par 3 Contest with family and dear friends Gary Player and Tom Watson!"
Matt Wallace won the Par-3 Contest by acing the eighth hole and beating Sandy Lyle in a three-hole playoff.
Nicklaus also participated in a "love letter" video for the Masters, looking back on his 60 years at Augusta National.
"I was just a teenager, taking in every moment of my first Masters. And at that time, I could never imagine all the wonderful, personal moments that would play out here and moments I cherish today."
You can live stream the 2019 Masters at Masters.com and CBSSports.com. Live TV coverage of the first round begins at 3 p.m. EDT on ESPN.