A person was filmed setting the mosque in Fort Pierce ablaze early on Monday morning, Florida officials said.
Omar Mateen committed the worst mass shooting in modern American history in June when he opened fire in Pulse, injuring 53 people.
He was killed by police after taking hostages and declaring his allegiance to the Islamic State group.
The fire at the Islamic Center of Fort Pierce may have been timed to coincide with the Muslim holiday Eid al-Adha and the anniversary of the 9/11 attacks.
St Lucie County Sheriff`s Office condemned the fire in a Facebook post, saying: "A fire at any place of worship is alarming, regardless of the circumstances."
A spokesman for the sheriff`s office, Major David Thompson, said in an early morning press conference that "this is a horrible tragedy. Not only for the Islamic Center, but for our community".
Multiple law enforcement agencies are investigating, including the FBI.
The Islamic Center of Fort Pierce has experienced other trouble in recent months. A few weeks after the nightclub shooting, a man was badly beaten outside the mosque.
Motorists frequently stop to shout abuse at worshippers, including the driver of a truck who stopped to shout insults earlier that same day, according to the Council on American-Islamic Relations.
In addition to Omar Mateen, the mosque was also visited by Moner Mohammad Abu Salha, who became the first American to commit a suicide bombing attack in Syria.
Orlando nightclub shooting: Fire set at gunman`s mosque
13 September 2016
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The mosque attended by the Pulse nightclub gunman, who killed 49 people in Orlando, has been set on fire.
