Tunicamycins with different fatty acid side chains were isolated from the culture broth of Bacillus cereus K-279, a strain isolated from a soil sample in Kawauchi-cho, Tokushima City. This is the first report of tunicamycin production by a bacterium, as the antibiotic was previously isolated only from Streptomycetes. Strain K-279 was taxonomically identified as Bacillus cereus based on characteristics including facultative anaerobic rods, motile, endospore formation, Gram-positive, catalase positive, and biochemical traits (e.g., acid from glucose, no acid from arabinose/xylose/mannitol). Fermentation was carried out in a seed medium (1% polypeptone, 0.5% meat extract, 0.5% NaCl, pH 7.0) at 28°C for 18hr, followed by transfer to a production medium (3% soluble starch, 0.5% glucose, 1% polypeptone S, minerals, pH 7.0) in a 30-l jar fermentor (28°C, 30hr, aeration 20 l/min, agitation 300 rpm), with maximum tunicamycin activity (2μg/ml) at 30hr. The active substance was purified via centrifugation, Diaion HP-20 column chromatography (eluted with alkaline methanol), n-butanol extraction, silicic acid chromatography, and HPLC, yielding four pure homologues (E, F, G, K) from 40 l of culture filtrate (60mg tunicamycin complex total). Structural confirmation via UV, IR, and NMR spectra confirmed the antibiotic as tunicamycin, with fatty acid side chains analyzed by GC-MS (after hydrolysis and methyl esterification); homologue G was a new tunicamycin with a 12-methyl-tridecanoic acid side chain. Antimicrobial activity tests (serial agar dilution) showed tunicamycin was active against Gram-positive bacteria (e.g., Bacillus species) and yeasts (e.g., Saccharomyces cerevisiae), with longer fatty acid chains and α,β-unsaturated side chains enhancing activity.