Six sesquiterpenoids 1-6, including two new ones, an ent-daucane-type sesquiterpenoid, asperaculane A (1), and a nordaucane one, asperaculane B (2), and four known nordaucane derivatives, aculenes A-D 3-6, together with the known secalonic acid D (7), were isolated from a fermentation culture of the fungus Aspergillus aculeatus. Their structures and absolute configurations were established by analyses of their spectroscopic data, including 1D and 2D-NMR spectra, HR-ESIMS, electronic circular dichroism (ECD) data, and quantum chemical calculations. These metabolites were evaluated for in vitro cytotoxic activity against two cell lines, human cancer cell lines (HeLa) and one normal hamster cell line (CHO).