The Central Committee of Sudanese Doctors, a medical group linked to protesters, said at least two people were killed and several wounded in the Monday morning raid, which was still in progress.
In a post on Twitter, the Sudanese Professionals Association (SPA) said the country's ruling transitional military council had assigned a large number of troops to disperse the protest camp.
The sound of heavy gunfire was heard in footage broadcast from the scene, while plumes of smoke rose into the sky.
Speaking to Al Jazeera, witnesses spoke of soldiers being heavily armed and of using live fear and tear gas against protesters who were peaceful and did nothing to provoke the troops.
"We are being attacked by the Rapid Support Forces and the police," Mamadou Abozeid, a protester in Khartoum, told Al Jazeera over the phone.
Mohammed Elmunir, another protester in the Sudanese capital, said security forces first blocked the exits of the sit-in site before opening fire on protesters.
"They were shooting at every one randomly and people were running for their lives. They blocked all roads and most tents at the sit-in have been set on fire," Elmunir told Al Jazeera.
"People are very angry right now. People don't know what could happen next. Protesters have disperse to other parts of the city. They are now on the streets protesting. Most people have blocked the roads in their neighborhoods." He added.
In a statement, the SPA said the ruling military council would be held accountable for any bloodshed and called for a campaign of civil disobedience.
"It is imperative to go out to the streets to protect the revolution and the remaining dignity. Our weapons are peace courage," it said in a statement.
"We call on the revolutionaries in all neighborhoods, villages, towns and cities of Sudan to go out to the streets and start marching, closing all streets and bridges and ports. We call for a comprehensive civil disobedience to bring down the deadly military junta and complete our revolution."
Nawal Osman, a protester in Khartoum, said she and her husband were getting ready to leave their house and join the protesters.
"I am going there now, I live nearby the protest area," she told Al Jazeera over the phone. "This revolution is staying," she added. "We are all ready to be there, to protect the country and to get rid of those criminals."
Sudanese human rights activist, Azaz Elshami, said it was not surprising the military has resorted to using deadly force to deal with protesters.
"What is happening is the mask is finally falling. The military is not different than the government before it. The are not what they said they are. They don't want change and they want power for themselves. Now, everyone knows what they are up against. This is a new phase. it might take much longer but I dont think Sudanese people will back down." Elshami told Al Jazeera.
The sit-in has become the focal point of Sudan's protest movement, which saw longtime autocrat Omar al-Bashir overthrown in April and has since been calling for the generals who replaced him to hand over power to a civilian-led administration.
The operation came days after Sudan's military rulers calledthe sit-in outside the defence ministry "a danger" to the country's national security and warned that action would be taken against what they called "unruly elements".
The military has also ordered the office of the Al Jazeera Media Network in Khartoum to be shut down, without giving any reason.
It also withdrew the work permits for the correspondents and staff of the Qatar-based news organization.
"People have started putting up barricades in many areas and it seems that the situation is aggravating," Hamid Eldood, a professor at Al Neelain University, told Al Jazeera from Khartoum.
He added that the latest developments indicated a "tone change" by the members of the transitional military council following the visit by the council's head, General Abdel Fattah al-Burhan, and his deputy, Mohamed Hamdan Dagalo - also known as Hemeti - "to Egypt, Saudi Arabia and the United Arab Emirates".
"It is really a barbaric act and it seems Sudan is heading towards civil war and a [bleak] future," Eldood said.
Source: Al Jazeera