The pontiff condemned the killings as "homicidal folly and senseless hatred" while Obama described them as "an act of terror and an act of hate".
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According to a tweet from the British royal family's official account, the queen was "shocked" by the attack, adding that the monarch and her husband's "thoughts & prayers are with all those who have been affected".
The attack in which a gunman opened fire at a packed nightclub in Orlando, Florida, is the worst mass shooting in US history.
Leaders from countries shaken by recent jihadist attacks, including France and Belgium, were among the first to express solidarity with the US.
Afghan President Ashraf Ghani sent his condolences to the families of the victims as authorities identified the alleged gunman as 29-year-old Omar Mateen, a US citizen of Afghan descent.
"I unequivocally condemn the horrific attack in Orlando, Florida, USA. Nothing can justify killing of civilians," Ghani tweeted.
Reacting to the latest in a litany of mass gun killings in the US, Obama said the FBI was "appropriately investigating this as an act of terrorism".
"Although it's still early in the investigation, we know enough to say that this was an act of terror and an act of hate," he said, later ordering flags at half-staff as an act of mourning.
Caleb, right, wipes tears as he stands with his partner Yosniel during a vigil in memory of the victims of the Orlando mass shooting, in Miami Beach. Source: AP
In a statement from the Holy See, the pope lamented the tragic loss of life.
The attack had "caused in Pope Francis, and in all of us, the deepest feelings of horror and condemnation... before this new manifestation of homicidal folly and senseless hatred", said the statement.
"We all hope that ways may be found, as soon as possible, to effectively identify and contrast the causes of such terrible and absurd violence," it added.
Source: AFP
'Barbaric crime'
In France, which remains on edge after Islamic State group jihadists killed 130 people in Paris in November 2015, President Francois Hollande reacted "with horror".
The president "expresses the full support of France and the French people to the US authorities and the American people during this ordeal", a statement from the Elysee Palace said.
Prime Minister Manuel Valls also expressed his "compassion" and "solidarity" with the American people.
"By striking the gay community, this terrible Orlando attack reaches us all," he wrote in French on Twitter.
Belgium's Prime Minister Charles Michel tweeted his condolences over the attack, which comes two-and-and-half months after dual strikes by jihadists on Brussels airport and metro that killed 32 people.
"Deeply saddened by the loss of so many innocent lives in Orlando shooting. We join families in their grief," he said.
In Britain, Prime Minister David Cameron said on Twitter he was "horrified" by the shooting. "My thoughts are with the victims and their families," he said
Israeli Prime Minister Benjamin Netanyahu, whose country saw four people shot dead by two Palestinian gunmen on Wednesday, also described the killings as "horrific".
"On behalf of the people and government of Israel I extend our deepest condolences to the American people following last night's horrific attack on the LGBT community in Orlando," he said in a statement issued in English.
Scenes from outside Pulse nightclub moments after the fatal shooting occurred. Source: EPA
"Israel stands shoulder to shoulder with the United States at this moment of tragic loss," Netanyahu added.
Russian President Vladimir Putin, meanwhile in a Kremlin statement, called the killings "a barbaric crime".
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