Three diphenyl ethers (<b>1</b>-<b>3</b>) and a cyclopentenone (<b>4</b>), together with seven known compounds (<b>5</b>-<b>11</b>), were isolated from the fermentation broth of the marine sediment-derived fungus <i>Spiromastix</i> sp. SCSIO F190. Compounds <b>3</b> and <b>4</b> were found to exist as a pair of atropisomers (<b>3a</b>, <b>3b</b>) and racemates (<b>4a</b>, <b>4b</b>), respectively. The planar structures of compounds <b>1</b>-<b>4</b> were elucidated on the basis of NMR and HRESIMS data sets. The absolute configurations of <b>2</b> and <b>3</b> were determined by spectroscopic and single-crystal X-ray diffraction analyses, whereas the configuration of <b>4</b> was determined by spectroscopic and chiral analyses. All compounds, except for <b>4</b> and <b>11</b>, displayed activities against various pathogenic bacteria. Notably, compounds <b>1</b>-<b>4</b>, especially <b>1</b>, exhibited strong activity against Gram-positive bacteria, including methicillin-resistant bacterial strains of <i>Staphylococcus aureus</i> (MRSA), <i>Enterococcus faecalis</i> ATCC 29212, and <i>Bacillus subtilis</i> BS01, with MIC values ranging from 0.5 to 4 μg/mL. Moreover, the structure-activity relationship analyses of the active compounds and their analogues revealed the critical structural features correlating to the observed antimicrobial activities, herein providing insights for antimicrobial drug development.