About 15,000 Gold Fields workers could be fired if they do not return to work by 2pm on Thursday, CEO Nick Holland said.
"We are now in the unfortunate position where we have exhausted all reasonable and legal alternatives," he said in a conference call on Tuesday.
"We are now left with no option but... to protect people, assets, and business."
The ultimatum applies to illegally striking workers at Gold Field's Beatrix one, two, and three shafts in the Free State and the KDC West mine near Carletonville in Gauteng.
Workers at Beatrix one, two, and three shafts started returning to work on Tuesday morning.
They had been on a wildcat strike since September 24.
Workers at KDC West have been on an illegal strike since September 9.
The ultimatum did not apply to the KDC East mineworkers who went on strike on Sunday.
Those workers who returned to work by the deadline would benefit from an agreement reached under the auspices of the Chamber of Mines "notwithstanding that the offer has expired", Holland said.
Gold Fields had lost about R1.2 billion in revenue and 65,000 ounces in gold production from the strike action.
The company had saved R900 million in unpaid wages over the same time period, Holland said.