Joseph Michael Schreiber faces at least 30 years in prison if convicted on charges of arson with a hate crime enhancement, said authorities.
The suspect allegedly made multiple anti-Muslim posts on social media.
The Islamic Center of Fort Pierce was heavily damaged in the fire, which was set on the anniversary of 9/11.
Omar Mateen, who occasionally went to the mosque, shot dead 49 people in Pulse nightclub in June before being killed.
He professed allegiance to the Islamic State group.
On Wednesday evening, St Lucie County Sheriff's Office said Mr Schreiber owns a motorcycle consistent with one shown on surveillance video footage outside the mosque on the night of the fire.
"Other evidence collected also linked Schreiber to the arson," Maj David Thompson told a news conference.
Schreiber has made a number of comments against Islam on what appears to be his Facebook page.
One post said: "ALL ISLAM IS RADICAL , and should be considered TERRORIST AND CRIMANALS and all hoo participate in such activity should be found guilty of WAR CRIM".
Mr Schreiber was previously sentenced twice to state prison for theft, according to the Florida Department of Corrections records.
He served his first term from 2008-09 and his second stretch from 2010-14.
No one was injured in the weekend's blaze, which coincided with the Muslim holiday Eid al-Adha.
The FBI and the Federal Bureau of Alcohol, Tobacco, Firearms and Explosives joined the investigation.
The blaze has left the mosque's members "saddened and scared", said assistant imam Hamaad Rahman.
In addition to Mateen, the mosque was also visited by Moner Mohammad Abu Salha, who became the first American to commit a suicide bombing attack in Syria.
A 32-year-old man has been arrested over last Sunday's fire attack on a Florida mosque, which was attended by the Orlando nightclub gunman.