Triene antibiotics containing β-lactone-γ-lactam bicyclospiro and oxazole rings are unique metabolites produced by Streptomyces spp., such as oxazolomycins and curromycins. In the course of continuing search for novel antimicrobial agents from microorganisms, an actinomycete strain Streptomyces sp. isolated from a soil sample collected in Taejon, Korea, was found to produce a new antibacterial compound 16-methyloxazolomycin (1). This paper describes the production, isolation, structural elucidation and biological activities of 1. The strain was cultured in seed medium and production medium, and 1 was isolated via centrifugation, 70% aqueous acetone extraction, Diaion HP-20 column chromatography, Kupchan's partition, ODS flash chromatography, Sephadex LH-20 gel filtration and reversed-phase HPLC, together with known oxazolomycin (2). Structural elucidation using ESI-MS, HRESI-MS, ¹H NMR, ¹³C NMR, HMBC and NOESY experiments revealed that 1 belongs to the oxazolomycin class, with a methyl group at C16 and S configuration at C16. 16-Methyloxazolomycin (1) showed antibacterial activity against Bacillus subtilis 1069 (MIC 5.0 μg/ml) and antialgal activity against Chlorella vulgaris IFO 15941 (MIC 10 μg/ml), as well as cytotoxicities against P388 leukemia cells (IC50 0.23 μg/ml) and A-549 human lung adenocarcinoma cells (IC50 4.6 μg/ml).