Noor Salman, the wife of Omar Mateen, was arrested by the FBI on Monday.
Mateen was responsible for the shooting rampage at Pulse nightclub in Orlando on June 12, 2016, which left 49 people dead. He pledged allegiance to the Islamic State group during a standoff with police.
Salman was arrested at her home near San Francisco in connection with the mass shooting.
Salman faces charges of obstruction of justice and aiding and abetting the attempted provision of material support to a foreign terrorist organization.
Salman was booked into Santa Rita Jail in Almeda County in California. She is expected to face a judge in Oakland on Tuesday morning.
Last year, Salman told The New York Times that she was aware her husband watched Jihadist videos, but she didn't think much of it because he had been cleared by the FBI.
"I am glad to see that Omar Mateen's wife has been charged with aiding her husband in the commission of the brual attack on the Pulse nightclub," Orlando Police Chief John Mina said in a statement. "Federal authorities have been working tirelessly on this case for more than seven months, and we are grateful that they have seen to it that some measure of justice will be served in this act of terror that has affected our community so deeply."
WFTV legal analyst Bill Sheaffer said in a 2016 interview about Salman that "in order to successfully prosecute the wife, they're going to have to show she did something in furtherance of that crime."
Salman will be transferred to the Middle District of Florida, the FBI said.
"I hope today's news provides some comfort to the families who are mourning their loved ones," Florida Gov. Rick Scott said in a statement.