Manuel Garcia Viejo died Thursday after his condition deteriorated, Xinhua reported.
The 69-year-old had arrived at the Carlos III Hospital in north of Madrid Monday.
His condition was described as serious as he was suffering from severe dehydration and his liver and kidney were affected due to the virus.
He was diagnosed with Ebola last Friday.
Viejo is the second Spaniard to pass away after 75-year-old Miguel Pajares died Aug 13 after six days of treatment.
In contrast to Pajares, Viejo did not receive the experimental ZMapp serum, which has produced positive results in some cases, because stocks had run out and although the hospital was looking for alternative treatments, he died before any could be applied.
Viejo had spent the past 12 years as medical director of the San Juan de Dios Hospital in the city of Lunsar in Sierra Leone.