Bacillaene, a novel polyene antibiotic (hexaene), was discovered and isolated from fermentation broths of a strain of Bacillus subtilis. The fermentation broth also contained the previously described antibiotics difficidin and oxydifficidin. Bacillaene has a nominal molecular weight of 580 and an empirical formula of C35H48O7. In in vitro agar diffusion assays, it inhibits both Gram-positive and Gram-negative bacteria but shows no inhibitory activity against Candida species, with especially potent activity against hyper-permeable strains of Escherichia coli. Studies in vitro indicate that the antibiotic inhibits prokaryotic protein synthesis but not eukaryotic protein synthesis and acts as a bacteriostatic agent.