A new cyclohexenone, phomaligol J (1), along with nine known compounds (2-10) were isolated from the liquid culture of the pufferfish-associated Aspergillus austwickii collected from Yangma Island, Yantai. Their structures were determined by extensive spectroscopic data analysis, high resolution electrospray ionization mass spectrometry (HRESIMS), electronic circular dichroism (ECD), and the comparison with reported data in the literature. In bioactivity, all isolated compounds were evaluated for antibacterial activity against 12 bacterial strains. Notably, compound 1 exhibited moderate antibacterial activity against Staphylococcus aureus with a minimum inhibitory concentration (MIC) value of 40 µg/mL.