One new oxygenated steroid, named aspersteroid A (1), and three known analogues (2-4) were isolated from the marine-derived fungus Aspergillus flavus collected from the Bohai Sea. Their structures were elucidated by spectroscopic analyses and by comparison with previously reported data. The absolute configuration of tetracyclic nucleus in 1 was assigned by quantum chemical calculation of the electronic circular dichroism (ECD) spectrum. All the compounds were evaluated for their cytotoxic and antibacterial activities.