He used his Twitter page to send the message just hours before the Opening Ceremony gets underway at the Maracana Stadium.
Good luck to the athletes at #Rio2016! May you always be messengers of goodwill and true sporting spirit.
— Pope Francis (@Pontifex) August 5, 2016
The 31st Summer Games will see billions tune in to watch 206 nations compete over the next two weeks.
Rio de Janeiro will hope to put months of bad press over doping, Zika virus and last-minute building works behind it as the competition gets underway.
Speaking from the Brazilian capital, Tom Ellis from Premier's media partner Passion for Sport told the News Hour that the atmosphere was building nicely.
He said: "It's starting to grow. It's very different to the [football] World Cup two years ago when you had big groups of fans congregating.
"This is a more chilled out atmosphere. People are looking forward to it, lots of different nationalities mixing and getting excited generally about the Games.
Before the Games get under way it is the host nation's turn to take centre stage as world watches the opening ceremony that will showcase its history, diversity and music.
Ellis says the city is perfect for an occasion of this kind.
"Rio is a great place for a sporting occasion, a celebration. They do like a party so the atmosphere is very colourful over here," he explained.
"Brazil has been in a difficult time with political and economic woes but people are starting to come together and this will hopefully bring people from all over the world together."
Andy Murray will lead Team GB's 366 athletes into the cavernous Maracana stadium as the flag bearer, describing the honour as "by far the proudest moment" in his career.
He'll be in action over the weekend as he looks to win Gold once again after his victory at Wimbledon four years ago.
Meanwhile the torch relay, which finishes at the Opening Ceremony, has been marred by protesters.
Riot police have used tear gas to disperse the crowds campaigning over workers' rights.
On Friday, Rio de Janeiro's Archbishop Orani Joao Tempesta held the torch in front of the Christ the Redeemer statue.
Listen to Tom Ellis speaking to Premier's Aaron James on the News Hour: