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Four new steroids, dendronecholones A–D (1 – 4), and two known analogues, 12 β ,16 β ,20-trihydroxycholesta-1,4-dien-3-one 16-acetate (5) and nanjiol A (6), were isolated from Dendronephthya soft coral collected in waters off Zhejiang Province, China. The structures were elucidated by extensive spectrometric and spectroscopic analyses involving 1D and 2D NMR experiments, high-resolution electrospray ionization mass spectrometry (HRESIMS), and single-crystal X-ray diffraction. Bioassay of the six compounds was performed on thirteen pathogenic Vibrios (members of the family Vibrionaceae), nine of which were directly isolated from the gut of a half-smooth tongue sole (Cynoglossus semilaevis) and black scraper (Navodon septentrionalis) after its death from bacterial infection. These anti- Vibrio tests indicated that compounds 3 and 6 displayed inhibition towards Vibrio parahaemolyticus with minimum inhibitory concentrations (MIC) of 8 μg/mL, compounds 1 – 6 exhibited inhibition against V. scophthalmi , with MIC values ranging from 8 to 32 μg/mL, compounds 1 , 2 and 6 also inhibited the growth of V. harveyi , with values of 32, 8 and 8 μg/mL. Dendronephthya soft corals are an unexplored biological resource for the development of these and other new anti- Vibrio lead compounds and potential aquaculture treatments. [Display omitted] • soft coral; Dendronephthya sp.; steroids; aquatic disease; anti- Vibrio activity [ABSTRACT FROM AUTHOR]