Mesophotic coral ecosystems (MCEs) represent an underexplored source of intriguing natural products. Efforts to discover bioactive metabolites from sponge-associated fungi in MCEs identified a new steroid, acremocholone (1) and its three known analogs (2–4), from Acremonium sp. NBUF150. The Acremonium sp. NBUF150 was isolated from a Ciocalypta sponge located 70 m deep within the South China Sea. The planar structures and absolute configuration of 1–4 were determined from NMR-derived spectroscopic data, HR-ESI-MS, and X-ray crystallography. Compound 1 exhibited antimicrobial inhibition against Vibrio scophthalmi, V. shilonii and V. brasiliensis at minimum inhibitory concentrations of 8 μg/mL; compound 2 inhibited V. shilonii and V. brasiliensis at 8 and 32 μg/mL, respectively, and compound 4 inhibited growth of V. brasiliensis at 16 μg/mL. Sponge associated fungi from MCEs represent a promising resource of anti-Vibrio drug leads for aquaculture use.