The 20-year-old singer’s comments come after the US court decided to overturn the landmark 1973 Roe v Wade case that legalised abortion nationwide.
During her set on the Pyramid Stage on Friday night, she spoke out against the ruling as she introduced her song Your Power, which explores people abusing their power.
Seated alongside her brother and co-writer, Finneas O’Connell, she said: “The song we’re about to do is, I think, one of the favourites that we’ve written and it’s about the concept of power and how we need to always remember how not to abuse it.
“And today is a really, really dark day for women in the US. I’m just going to say that as I cannot bear to think about it any longer in this moment.”
This appearance was particularly poignant for the singer as she became Glastonbury Festival’s youngest ever solo headliner.
During the electrifying set, she treated the audience of adoring fans to a selection of hit songs from her two number-one albums, including Bury A Friend, When The Party’s Over, NDA and My Strange Addiction.
She gave a typically energetic performance, running and dancing across the stage, wearing a black and white graphic outfit with black striped arm-warmers and her hair in space buns.
The singer delivered another political moment during the set as she spoke of how society needed to better protect the environment.
Ahead of performing Everything I Wanted, she said: “So I think we all know that we really need to do a better job of protecting the planet that we all live on.
“We always can do a better job of protecting our people and our friends and family and strangers and standing up for each other and standing with each other.
“And I love you all and I stand with you and I support you and everything that makes you who you are.”
The audience appeared to reciprocate back her affection as throughout the set they sang, danced and chanted her name.
Near the end of her set she admitted she felt “so undeserving” of this opportunity as she thanked the festival and the fans for having her.
She added: “I am so honoured to be here among all of you and all the incredible artists and the history of this festival.”
Eilish closed her history-making show with an explosive rendition of her hit track Happier Than Ever.
The singer energised the crowd as she delivered the dramatic chorus line which had the crowd screaming the lyrics alongside her.
This marks the US pop star’s second appearance at Glastonbury after making her debut in 2019 when she performed on the Other Stage.
Glastonbury continues throughout the weekend.