The self-proclaimed Islamic State, or IS, has claimed responsibility for the massacre, the deadliest attack on US soil since the September 11 attacks of 2001.
The Federal Bureau of Investigation says it is likely the attacker was lured to groups such as IS at least in part through the internet.
A series of Snapchat videos was shared by an Orlando nightclub reveller shortly before the massacre, her last post capturing some of the sound of gunfire.
The 25 year-old woman who recorded the video was among the 49 people killed.
At the bullet-ridden scene, investigators have been poring over evidence which could explain the motives of attacker Omar Mateen.
The 29 year-old son of Afghan migrants called emergency services during the shooting and pledged allegiance to IS, also known as ISIL or ISIS.
But FBI director James Comey says it is not entirely clear which militant group the gunman aspired to support.
"During the calls, he said he was doing this for the leader of ISIL, who he named and pledged loyalty to. But he also appeared to claim solidarity with the perpetrators of the Boston Marathon bombing and solidarity with a Florida man who died as a suicide bomber in Syria for al-Nusra Front, a group in conflict with the so-called Islamic State."
The attack has reignited debate over how best to confront such threats and become a point of disagreement in the campaign for the U-S presidential election.
Presumptive Republican nominee Donald Trump has repeated his call for a temporary ban on Muslims entering the country.
"It will be lifted, this ban, when and, as a nation, we are in a position to properly and perfectly screen these people coming into our country. They are pouring in, and we don't know what we're doing."
Presumptive Democratic candidate Hillary Clinton, in her first campaign stop since the shootings, says the country should go carefully in bolstering security without demonising Muslims.
She also called for tougher gun-safety measures and increased efforts to remove IS messages from the internet.
"We have to do a better job intercepting ISIS's communication, tracking and analysing social-media posts and mapping jihadist networks, as well as promoting credible voices who can provide alternatives to radicalisation."
US president Barack Obama says he is concerned the response to the massacre could turn into a debate over whether to toughen gun laws or to pursue groups such as IS.
"The suggestion is either we think about something as terrorism and we ignore the problems with easy access to firearms, or it's all about firearms and we ignore the role, the very real role, that organizations like ISIL have in generating extremist views inside this country. And it's not an either/or. It's a both/and."
In Orlando, mayor Buddy Dyer has praised the community for the way it has come together to support the family and friends of the victims.
And he says the city will heal.
"We will not be defined by the act of a cowardly hater. We will be defined by how we respond, how we treat each other, and this community has already stepped up to do that."
