The 75-year-old Roman Catholic priest, Miguel Pajares, was being treated in Spain with an experimental US serum, ZMapp, after being flown to Madrid on August 7 - the first patient to be evacuated to Europe in the fast-spreading African outbreak.
He contracted Ebola at the Saint Joseph Hospital in the Liberian capital Monrovia where he worked with patients suffering from the disease.
"It is confirmed. He died at 9:28 am (0728 GMT)," said a spokeswoman for the Spanish capital's La Paz-Carlos III hospital, confirming that he had been treated with ZMapp.
"We hoped he would be able to overcome the disease but it was not to be. It is as God wished," the priest's sister-in- law Carmen Romo told Spanish radio station COPE.
Ebola has claimed four lives in 10 days among the staff at Saint Joseph Hospital in Monrovia, including its director, Cameroon-born Patrick Nshamdze. The hospital was closed on August 1.
A Roman Catholic order that set up the charity running the hospital has said it fears inadequate safety precautions were put in place.
Its spokeswoman, Adriana Castro, told AFP yesterday that an earlier test had wrongly shown the late director of the Liberian hospital to be free of Ebola.
"What we fear is that as Patrick's test showed negative, many of them relaxed," the spokeswoman said.
"We don't know 100 per cent but it is probable that is how Pajares was infected and possibly from Pajares the virus extended among people who were there."
"Until they knew they were positive with Ebola they did not take precautions," she said.
The latest outbreak - which the WHO says is by far the worst since Ebola was discovered four decades ago - has killed over 1,000 people and around 55-60 per cent of those infected.
The Spanish religious order declined to comment on letters purportedly from Pajares describing his fear of Ebola and a lack of basic equipment to protect against its spread.
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