Officials say the nurse died Tuesday after treating a man who arrived from Guinea at a clinic in the capital Bamako.
The patient was suffering from kidney failure and later died, medical sources said late Tuesday, and authorities now believe he also had Ebola, although this has not been confirmed.
The Bamako clinic is now in quarantine.
The case -- involving the first Ebola death of a Malian national in the country -- has raised fears of further contamination as it was unrelated to Mali's only other confirmed fatality, that of a two-year-old girl.
Almost 5,000 people have been killed by Ebola in the west African outbreak according to official data from the World Health Organization (WHO), which says the true scale of the epidemic could be much greater.
The virus kills around 70 percent of its victims, often shutting down their organs and causing unstoppable bleeding.
The new case in Mali came a day after the WHO said it had released from isolation 25 of more than 100 people thought to have come into contact with country's first victim.
Two-year-old Fanta Conte died after returning from a trip to Guinea. The infection sparked panic as the toddler had travelled by bus and taxi with her grandmother, sister and uncle, making frequent stops on a trip of more than 1,200 kilometres (750 miles).
They also spent two hours in Bamako, visiting relatives in a house of 25 people.
Morocco was stripped of hosting football's 2015 Africa Cup of Nations on Tuesday and flung out of the competition after insisting that it wanted to postpone the tournament due to fears over the virus.
Meanwhile, the virus is continuing to spread in Sierra Leone, where the WHO said cases were "still skyrocketing" in the west of the country, including the capital Freetown.