Prime Minister Malcolm Turnbull has extended his "deepest sympathies" to those impacted by the massacre at a gay nightclub in the US city of Orlando, describing the attack as an "assault on freedom".
At least 50 people have been killed and more than 50 injured in the attack that took place on Sunday night Australian time.
Omar Mateen, 29, has been identified as the shooter.
Speaking on Monday morning, Turnbull said the killer was likely motivated by hatred.
"It's too early to say precisely what motivated the gunman but it certainly appears to be motivated by a hatred of the freedoms, the free society in which all of us enjoy," he said.
"An attack like this, which has killed at least 50 people and injured as many more, is not simply an assault on the people that have been killed and injured, it's an assault on every one of us.
"It's an assault on freedom. I want to say to Australians that we convey our deepest sympathy and condolences to the families of those who are injured and I will be conveying those more formally to the President in due course."
Opposition leader Bill Shorten said he was shocked and saddened by what had occurred in Florida.
"Australia grieves with the loved ones of the dead. Our thoughts too are with the injured. These murders are despicable, and cowardly. We condemn them, utterly," he said in a statement.
"Many in the LGBTI community will feel an added layer of pain and a sharper sense of loss in the wake of this tragedy. We stand with you in solidarity."
Greens leader Richard Di Natale called the mass shooting senseless.
"Violence and hate have no place in our communities and now more than ever we must stand together against these hateful acts," he said.
Earlier, Barack Obama described the attack as an act of terror.
"We know enough to say this was an act of terror and an act of hate," he said.
"The FBI is appropriately investigating this as an act of terror. We will go wherever the facts lead us ... What is clear is he was a person filled with hatred."
Obama said the massacre was "especially heartbreaking day for our friends who are lesbian, gay, bisexual or transgender".
The attack is the deadliest mass shooting in American history.