In what has become an annual tradition, Mr Eavis took to the stage at Croissant Neuf, this year choosing The King’s classic Can’t Help Falling In Love, followed by Frank Sinatra’s My Way.
Earlier on Thursday, thousands of voices rang out across Worthy Farm as campers joined in song to pay tribute to murdered MP Jo Cox.
The event had originally been scheduled for Wednesday, which would have been Jo Cox’s 42nd birthday, but was pushed back after many were delayed arriving at the site due to heavy traffic.
Singer Billy Bragg led attendees in a solemn version of We Shall Overcome as people spilled around the sides of the packed Park Stage to celebrate her life.
He was joined on stage by Emily Eavis, carrying her newborn baby, and more than a hundred women from the crowd who wore sashes in the purple, white and green of the Suffragettes.
A minute’s silence was then held for the Labour MP, who was killed on June 16.
The crowd joined in a march to The Sisterhood, the first women-only venue at the festival, which was officially opened in Ms Cox’s memory.
With the tribute event falling on the day of the EU referendum, Bragg told the crowd: “It’s a terrible irony that we are gathered together today on perhaps the most decisive day in the modern history of our country.
“Whatever happens tomorrow, whatever your politics are, tomorrow morning we will have to begin the process of healing in this country.
“We, all of us, must try to live up to the words of Jo Cox, who said whatever the divisions between us, we have much more in common than the things that divide us.
“Tomorrow we must live by those words.”
The late David Bowie’s life was also celebrated at the unofficial Glastonbowie, a singalong event which attracted thousands of fans – many dressed as the music legend himself.
In front of the Pyramid Stage, which is decorated with an Aladdin Sane lightening bolt in tribute to the late singer, fans gathered to sing Bowie’s hits including Starman, Ziggy Stardust and Suffragette City.
Organizer Miz said: “I started it a week after he passed away, and it was just to basically deal with my own sorrow. I’ve been a Bowie fan since I was a teenager myself and when he passed away it just felt like music died.
“I just thought I had to do something to bring some kind of joy to people. For people to keep singing Bowie songs and remembering him.”
With the music festival due to kick off at 11am on Friday, festival-goers got their first taste of the big-name acts as Rat Boy performed a secret gig at William’s Green.