Marine fungi are well known for their ability to produce a multitude of natural products and have been proved to be a particularly rich source of drug leads. Here, 20 pyrones and their analogs (1-20), including two new compounds (1and6), were obtained from a marine-derived fungus strain ofAspergillussp. DM94. Their structures were determined by analyses of UV, IR, HR-ESI-MS, and NMR data. The ability to inhibitHelicobacter pyloriin vitro was assessed for these isolated compounds. Results showed that thebis-naphtho-gamma-pyrones exhibited potent antibacterial activity against both the standard and multidrug-resistantH. pyloristrains. Structure-activity relationship (SAR) analysis suggested that thebis-naphtho[2,3-b]pyrones showed better anti-H. pyloriactivity than a hybrid of naphtho[2,3-b]pyrone and naphtho[1,2-b]pyrone. In addition, the free hydroxyl group of the C-8 position in the lower unit is vital for its anti-H. pyloriactivity. Importantly, compound18showed a synergistic effect in combination with amoxicillin, clarithromycin, or metronidazole, suggesting its potential use to overcome antibiotic resistance ofH. pylori. This study shed light on the discovery of new anti-H. pyloriagents.