Many recent studies have shown that peptic ulcer diseases are mainly caused by Helicobacter pylori (H. pylori) infection. Eradication of this bacterium dramatically decreases the recurrence rate in peptic ulcer patients. However, current treatment regimens including a proton pump inhibitor and antimicrobial agents such as amoxicillin and clarithromycin have problems including side effects (e.g. diarrhea), build-up of drug resistance, and poor compliance, so the development of a new class of anti-H. pylori agents is needed. In the course of screening for anti-H. pylori agents, N-acetyl aureothamine (1) was found from the culture broth of Streptomyces netropsis JCM4544. This paper describes the fermentation, isolation, structure elucidation and biological properties of 1, and also reports the anti-H. pylori activities of other γ-pyrone compounds. The molecular formula of 1 was determined to be C24H27NO5 by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and NMR data. The structure of 1 was elucidated through analysis of one- and two-dimensional NMR spectra (COSY, HMQC, HMBC) and NOESY. Antimicrobial activity tests showed that 1 exhibited potent anti-H. pylori activity with an MIC value of 0.003 μg/ml. Other γ-pyrone compounds, aureothin (2) and actinopyrone A (3), also showed potent anti-H. pylori activities, with 3 having 250-fold higher activity than amoxicillin and 130-fold higher than clarithromycin. Moreover, these compounds were inactive against other Gram-positive and Gram-negative bacteria tested, and had low in vitro cytotoxicity against HeLa S3 cells (IC50 values of 5.0, 10 and >10 μg/ml for 1, 2 and 3, respectively). These results suggest that γ-pyrone compounds are selective and potent anti-H. pylori agents with low potential for diarrhea caused by the disturbance of intestinal microbial flora.