Prosecutors have convened a grand jury to investigate Noor Salman, sources quoted by Fox News and Reuters say.
Noor Salman is reported to have told police she tried to talk her husband out of attacking the Pulse nightclub.
Orlando attack was the worst mass shooting in recent US history.
Fifty-three people were wounded and six remain in a critical condition.
Prosecutors quoted by Fox News said they were seeking to charge Noor Salman as an accessory to 49 counts of murder and 53 counts of attempted murder, as well as with failure to warn authorities about the impending attack.
It was possible that Omar Mateen had called his wife from inside the club while the killings were taking place, Fox quoted a source as saying.
Although Noor Salman has been questioned since the attack on June 12, she has not been arrested.
Senator Angus King, a member of the Senate Intelligence Committee which received a briefing on the investigation, told CNN that “it appears she had some knowledge of what was going on”.
He said: “She definitely is, I guess you would say, a person of interest right now and appears to be co-operating and can provide us with some important information.”
On June 14, media reported that Noor Salman had gone with Omar Mateen to buy ammunition and had also driven him to the Pulse nightclub on a previous occasion because he had wanted to survey it.
However, Noor Salman said she had tried to talk her husband out of carrying out the attack, sources quoted by NBC News said.
Omar Mateen’s father, Seddique Mateen, said on June 14 that Noor Salman – his son’s second wife – had returned to the couple’s flat on June 13 to pick up some clothes. He said she and the couple’s young son were still in Florida but declined to say where.
Omar Mateen, 29, who was killed when police stormed the club, pledged allegiance to ISIS during the attack, authorities say.
The FBI is investigating reports that Omar Mateen made several visits to the Pulse nightclub and made contact with other men on gay dating apps.
On June 14, President Barack Obama said Mateen appeared to have been “an angry, disturbed, unstable young man who became radicalized”.
The president also attacked a proposed ban on Muslims traveling to America from Republican presidential nominee Donald Trump as “not the America we want”.
Investigators have said there is no evidence that Omar Mateen had been in contact with any outside groups such as ISIS.
Omar Mateen was a US citizen, born in New York to Afghan immigrant parents.
Barack Obama will travel to the scene of the attack in Orlando on June 16.
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