Chastainbeat out fellow nominees Olivia Colman (The Lost Daughter), Penelope Cruz (Parallel Mothers), Nicole Kidman (Being the Ricardos) and Kristen Stewart (Spencer) to win the Oscar.
Sunday's award marked Chastain's first Oscar, after previously being nominated for The Help and Zero Dark Thirty. Earlier this awards season, the actress nabbed the Critics Choice Award and the Screen Actors Guild Award for her role.
"Thank you so much to the Academy for this," the 45-year-old actress said in her acceptance speech, before crediting her nominees. "I have to say your names -- Kristen, Nicole, Penelope, Olivia -- I love you guys so much. To be included in the conversation with you is such an honor. I have to thank Michael Showalter, my director, who created a space that inspired creativity and love and courage. And the incredible hair and makeup team... who really helped me find her."
"My scene partner and collaborator, Andrew Garfield, the brilliant Andrew Garfield. Thank you so much for bringing out the best in me by showing up with the best of you," Chastain added. "And right now we are coming out of some difficult times that have been filled with a lot of trauma and isolation. And so many people out there feel hopelessness and they feel alone. And suicide is a leading cause of death in the United States. It's touched many families. It's touched mine. And especially members of the LGBTQ community, who oftentimes feel out of place with their peers. We're faced with discriminatory and bigoted legislation that is sweeping our country with the only goal of further dividing us."
"There's violence and hate crimes being perpetuated on innocent civilians all over the world. And in times like this, I think of Tammy and I'm inspired by her radical acts of love," she said as she continued her speech. "We've talked about love a lot tonight. And I'm inspired by her compassion and I see it as a guiding principle that leads us forward. And it connects us all in the desire that we want to be accepted for who we are, accepted for who we love and to live a life without the fear of violence or terror. And for any of you out there who do in fact feel hopeless or alone, I just want you to know you are unconditionally loved for the uniqueness that is you."
Chastain also sent a message of love to her two children with husband Gian Luca Passi de Preposulo, whom she married in 2017. "You are my heart and I thank you all very, very much," she ended her speech.
The actress was superstitious ahead of the Oscars when she told ET's Nischelle Turner and Kevin Frazier she didn't believe her name would be called for Best Actress.
"These women are phenomenal," she told ET of her fellow nominees. Not believing she'd win, Chastain was happy to be there to celebrate her fellow nominees. "They're like my goddesses," she said, "so I'm so happy."
When ET spoke with Chastain about her Eyes of Tammy Faye role last September, she opened up about her transformation into the infamous and eponymous televangelist.
"I'm a perfectionist," Chastain told ET. "I mean, listen, I grew up watching her being made fun of [on late-night shows], and I guess the proximity of that made me think, 'OK, I could end up a late-night joke. Here we go!' What inspired me to go forward is Tammy did. She was like, 'People might laugh at me but I'm going to give them love.' I wanted to be inspired to do that."