The women who refused the intimate inspection were "assaulted and handcuffed using cable ties as if they are criminals", the National Union of Miners said in a statement that claimed 52 women were suspended.
Mine owner Sibanye Gold confirmed that it had suspended a number of female employees for "allegedly attempting to assist illegal miners", but put the figure at 45.
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"It is unacceptable and deplorable what these male security officers are doing at Sibanye Gold Cooke Operations," it added referring to the mine in Westonaria, southwest of Johannesburg.
"This is the worst violation of their human rights and degrading to their dignity."
Since January, 665 illegal miners have been arrested at the mine, and 123 employees have been suspended for smuggling food and other contraband to the illicit miners, the company said in a statement.
In June the NUM and Sibanye Gold signed a deal banning workers from taking food into the shafts in an effort to combat illegal mining.
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"Clothing is searched for food items in the presence of the employee and two protection services employees," Sibanye said.
"Female employees are searched by females and the male employees are searched by males."
Thousands of illegal miners operate in South Africa, often drawn from neighbouring Zimbabwe, Mozambique and Lesotho, risking their lives mostly in disused mines in the hope of retrieving small amounts of gold residue to sell on the black market.
Gold mining was South Africa's life-blood for centuries and helped the country become the most developed economy in Africa but production has dwindled in recent years as reserves are exhausted.
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