Two new steroid derivatives, 3-oxoergosta-4,6,8(14),22-tetraen-27-oic acid (1) and (14R,24R)-14-methyl-24,25-dihydroxycholesta-4,8-dien-3-one (2), together with one anthraquinone (3), one amino acid derivative (4), five cyclic dipeptides (5-9) and a quinazolinone alkaloid (10), were isolated from a deep-sea-derived Aspergillus versicolor SCSIO 41325. Notably, compound 2 features an unreported cholesta-4,8-dien-3-one steroid scaffold among natural products. The structures were determined by NMR and high-resolution electrospray ionization mass spectroscopy (HR-ESI-MS) spectroscopic methods, single-crystal x-ray diffraction measurements and comparison with literature data. All isolated compounds were subjected to a broad range of biological assays, including tests against pathogenic bacteria and fungi, as well as for inhibition of pancreatic lipase, acetylcholinesterase, neuraminidase, and antioxidant activity evaluations.