"We are still learning all the facts. This is an open investigation. We’ve reached no definitive judgment about the precise motivations of the killer," the president said. "What is clear is that he was a person filled with hatred."
The FBI is leading the probe in coordination with state officials, Obama said.
"We must spare no effort to determine what, if any, inspiration or association this killer might have had with terrorist groups," Obama said.
But Obama, who took no questions, also called for calm and urged Americans to pull together rather and live up to the country's defining values. After the attacks in Paris, Brussels and San Bernardino, Calif., some Republicans called for a temporary ban on Muslim immigrants or religious tests designed to prevent Muslim refugees from Iraq and Syria from coming to the United States.
Obama did not mention those proposals, but they were clearly on his mind as he called on Americans not to "give into fear or turn against each other."
Obama described the attack as "a sobering reminder that attacks on any American — regardless of race, ethnicity, religion or sexual orientation — is an attack on all of us and on the fundamental values of equality and dignity that define us as a country."
"And no act of hate or terror will ever change who we are or the values that make us Americans," he said.
He urged Americans to "stand united" and vowed to "take action against those who threaten us." Obama's tone was somber and betrayed little of the anger and frustration so apparent after other mass shootings.
The president made a point of noting that the nightclub "was a place of solidarity and empowerment," where members of the city's gay, lesbian, bisexual and transgender community "came together to be with friends, to dance and to sing and to live."
Noting that the killer used a handgun and an assault weapon in the attack, Obama reiterated his call for additional gun controls in America. Obama had made similar comments in the wake of other mass shootings, and he made no suggestions that what he called "the most deadly shooting in American history" would change the politics of gun control.
"This massacre is therefore a further reminder of how easy it is for someone to get their hands on a weapon that lets them shoot people in a school or in a house of worship or in a movie theater or in nightclub," he said. "And we have to decide if that’s the kind of country we want to be. And to actively do nothing is a decision as well."
Greg Jaffe contributed to this report.