7-Nor-ergosterolide (1), a rare 7-norsteroid with an unusual pentalactone B-ring system, and two new steroid derivatives, 3β,11α-dihydroxyergosta-8,24(28)-dien-7-one (2) and 3β-hydroxyergosta-8,24(28)-dien-7-one (3), were characterized from the culture extract of Aspergillus ochraceus EN-31, an endophytic fungus isolated from the marine brown alga Sargassum kjellmanianum. In addition, nine known related steroids were isolated and identified. The structures of these compounds were established on the basis of extensive spectroscopic analysis. The absolute configuration of the new steroids (1-3) was determined by application of the modified Mosher's method. Compound 1, which represents the first example of a 7-nor-ergosteroid possessing a pentalactone B-ring system in a naturally occurring steroid, displayed cytotoxicity against NCI-H460, SMMC-7721, and SW1990 cell lines with IC(50) values of 5.0, 7.0, and 28.0 μg/mL, respectively. Compound 2 also displayed cytotoxicity against the SMMC-7721 cell line with an IC(50) value of 28.0 μg/mL.