<p>Marine fungus-derived natural products are an important source of new drugs and lead compounds. In this study, guided by the Global Natural Products Social (GNPS) molecular networking strategy, four new cyclohexenone derivatives, aspergiflones A–D (<b>1</b>–<b>4</b>), together with three known analogues (<b>5</b>–<b>7</b>), were isolated from the coral-derived fungus <i>Aspergillus flavus.</i> The structures of these compounds were elucidated through extensive spectroscopic analyses, comparison with literature data, hydrolysis reactions, quantum chemical calculations, and TDDFT-ECD calculations. Structurally, aspergiflone A (<b>1</b>) is a novel cyclohexenone dimer featuring an unprecedented skeleton in which two cyclohexenone units are connected at C-15 via a C−C single bond. Compound <b>1</b> exhibited potent antibacterial activity against <i>Escherichia coli</i>. Mechanism studies revealed that compound <b>1</b> disrupts bacterial cell morphology, increases cell membrane permeability, and decreases intracellular K⁺ ion levels. Additionally, transcriptome analysis revealed that the differentially expressed genes (DEGs) were mainly associated with cellular process regulation, stimulus response, catalytic, and binding functions. Several pathways were also significantly affected in <i>Escherichia coli</i>, including ABC transporters, cofactor biosynthesis, energy metabolism, and arginine biosynthesis. The finding indicated that aspergiflone A has considerable potential in food safety.</p> Graphical abstract <p></p>