Miguel Pajares, 75, was airlifted to the Spanish capital from Liberia on August 7 after contracting the virus, which has already claimed the lives of over 1,000 people in the current epidemic sweeping West Africa.
He was struck down while working for a non-governmental organisation in the country.
He was repatriated to Spain alongside nun Juliana Bohi, who has tested negative for the disease.
But his death will raise fears that despite frantic efforts to contain the spread, its reach has spread beyond African shores.
Miguel was treated with a rare experimental drug created to treat the disease but it failed to save his life.
ZMapp has been engineered with three antibodies which bind to infected cells so the immune system can kill them.
The two American aid workers currently being treated at Emory University Hospital in Atlanta after being flown back to the US have also received doses of the drug.
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