Dr Samuel Brisbane was treating Ebola patients at the country's largest hospital, the John F Kennedy Memorial Medical Center in Monrovia, when he fell ill. He died Saturday, said Tolbert Nyenswah, an assistant health minister.
A Ugandan doctor died earlier this month.
The American, 33-year-old Dr Kent Brantly, was in Liberia helping to respond to the outbreak that has killed 129 people nationwide when he fell ill, according to the North Carolina-based medical charity, Samantha's Purse.
"We are hopeful, but he is certainly not out of the woods yet," she said.
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Early treatment improves a patient's chances of survival, and Strickland said Brantly recognised his own symptoms and began receiving care immediately.
There is no known cure for the highly contagious virus, which is one of the world's deadliest. At least 1,201 people have been infected in Liberia, Sierra Leone and Guinea, according to the World Health Organisation, and 672 have died.
Ominously, Nigerian authorities said Friday that a Liberian man died of Ebola after flying from Monrovia to Lagos via Lome, Togo. The case underscored the difficulty of preventing Ebola victims from traveling given weak screening systems and the fact that the initial symptoms of the disease including fever and sore throat resemble many other illnesses.
Health workers are among those at greatest risk of contracting the disease, which spreads through contact with bodily fluids.
The WHO says the disease is not contagious until a person begins to show symptoms. Brantly's wife and children had been living with him in Liberia but flew home to the US about a week ago, before the doctor started showing any signs of illness, Strickland said.