They released a video claiming that failures by the then-mayor of New York led to the deaths of 121 men.
The video says Mr Giuliani failed to provide working radios for crews and halted the recovery effort too soon.
His campaign denounced the footage, saying the union behind it was known to support Democratic candidates.
Mr Giuliani is seen as one of the frontrunners in the race for the Republican nomination.
'Taking the hit'
The 13-minute footage was made by the 280,000-strong International Association of Firefighters (IAFF).
It says Mr Giuliani pushed for a faster clean-up at Ground Zero before all the remains had been recovered, and placed an emergency centre in a building that later collapsed.
It also accuses the former mayor of failing to provide working radios for firefighters, saying it made it impossible for them to learn that the Twin Towers were about to collapse.
"Virtually the whole thing goes back to him with the radios," Jim Riches, a fireman whose son was killed on 9/11, says in the footage.
"He's the guy on the top, and he's the guy you yell at. He takes the hit. And my son is dead because of it."
The video - which also offers testimony from other firefighters and their relatives - says Mr Giuliani is exploiting the disaster as a theme for his presidential candidacy.
'Disgrace'
In response, Mr Giuliani's camp accused the IAFF - who has feuded often with Mr Giuliani since the attacks - of playing partisan politics.
Former firefighter Lee Ielpi, whose son died in the attacks, called the footage a "disgrace", dismissing the allegations against Mr Giuliani.
"I was there. I saw it. I experienced it. I'm not going to let lies like this go," he said.
Mr Giuliani's campaign also said he had a long history of supporting firefighters on health and safety issues.