Miguel Pajares, a 75-year-old Roman Catholic priest, tested positive for the disease at a hospital in the Liberian capital Monrovia where he worked, Spanish charity Juan Ciudad ONGD said in a statement.
He has worked in Liberia for over five decades, the last seven years at the Saint Joseph Hospital in Monrovia.
Pajares has been in quarantine at the hospital along with five other missionaries since the death on Saturday of its director from Ebola.
Two other women who were also in quarantine, one from Congo and the other from Guinea, also tested positive for Ebola, the aid organisation said.
During an interview broadcast on Monday, Pajares said he and the other missionaries in quarantine would like to be taken to Spain for treatment.
"I have a fever. I don't have any appetite, I could go without eating anything, I have a lot of pain in my joints. I need help to move from one place to the other," he told CNN en Espanol, a 24-hour Spanish-language news network.
"We hope that we can be evacuated. For us it would be a huge joy because if we are taken to Spain we would be in good hands and we could get better, God willing," he added.
Two Americans who worked for Christian aid agencies in Liberia and were infected with Ebola while taking care of patients in Monrovia were brought back to the United States for treatment in recent days.
Over 1,600 people who have been infected with the hemorrhagic fever in West Africa since March, marking the largest outbreak of Ebola in history.
A total of 887 people have died of the virus in Sierra Leone, Liberia, Guinea and Nigeria, a 55 percent fatality rate, according to the World Health Organisation.
The World Bank has pledged up to $200 million to Guinea, Liberia and Sierra Leone to help contain the outbreak.
Ebola is transmitted through close contact with bodily fluids, and people who live with or care for patients are most at risk.