The gunman was tackled by members of the Greater Faith Christian Church congregation and held for police, Orlando cable television station Central Florida News 13 reported. No others in the church were hurt.
The Polk County Sheriff's Office identified the suspect as Jeremiah Fogle, 57.
The St. Petersburg Times reported the dead woman was identified by family members as Fogle's 55-year-old wife Theresa.
Pastor William Boss and associate pastor Carl Stewart were transported to Lakeland Regional Medical Center. Sheriff Grady Judd said Boss was in the hospital's intensive care unit, the Times reported. Stewart, whose injuries were more serious, underwent surgery and had a tube placed in his chest, authorities said.
"We don't know exactly why he went into this mad rage," the Times quoted Judd as saying. "It's sad. Of all places where you should be safe, you should be safe in your house of worship."
The sheriff said Fogle allegedly fatally wounded his wife sometime before 10 a.m. at their home, then walked a block to the church, entered the front door of the sanctuary and walked down the center aisle as about 18 people were kneeling in prayer and the two ministers stood near the altar.
The sheriff said Fogle allegedly walked up behind Boss and shot him in the head with a .32-caliber revolver and then walked toward Stewart and shot him three times.
Parishioner Derrick Foster is credited with yanking the gun from Fogle while another worshiper, Corey Reed, hit the gunman with a microphone stand. The two men subdued Fogle and held him until deputies arrived.
"There's no doubt in our minds that Derrick Foster and Corey Reed are heroes," Judd said. "They stopped other people from being shot and the pastors from being shot more."
Authorities said the gun had one more bullet in it and Fogle had six more rounds in his pocket.
Fogle was a one-time deacon at the church, where he and wife were married in 2002, Judd said. He had left the congregation after a falling out with church leaders, the sheriff said.
The Times reported investigators said Fogle was arrested for first-degree murder in 1986 but it wasn't known if he was ever prosecuted.
Maria Beauford, 48, of Poinciana, the slain woman's sister, said the family didn't know what prompted the shootings.
"He always has a smile on his face; he's always glad to see everyone," she said of her brother-in-law.
She said the couple had formed their own outreach, Emmanuel Circle of Prayer Ministry, about three years ago, and owned an airport shuttle service.