"We are alarmed that we are even considering an offer from Walmart to take over key strategic national companies in South Africa," Cosatu said in a statement.
This followed the announcement on Monday that Walmart -- the largest retailer in the world -- had made a R30 billion offer to acquire JSE-listed Massmart.
"We will oppose the setting up of any Walmart stores in the Western Cape," the union said.
Walmart was "notoriously anti union" and an affront to workers' rights wherever it operated.
"We call for urgent national action from government to investigate this hostile move by Walmart.
"Companies whose practice it is to abuse workers rights are not welcome in South Africa."
Cosatu said the undermining of rights in one chain of stores would force non compliance in other chains of stores and put South Africa on a downward spiral in terms of labour law compliance.
"Walmart is not welcome in the Western Cape."
The global retail giant, however, vowed to respect the country's labour legislation.
"We respect and honour pre-existing union relationships and are committed to abiding by South African labour laws," Doug McMillon, president and CEO of Walmart International said in a statement on Monday.
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