In recent years, it has been recognized that bacteria can produce antibiotics of wide structural diversity, and many antibiotics known as products of Actinomycetales have been reported to be produced by eubacteria. Here, we report the isolation of azomycin from the culture broth of Pseudomonas fluorescens strain PB-6282, which was isolated from river water in Matsusaka-city, Mie Prefecture, and identified as P. fluorescens based on characteristics including aerobic, Gram-negative, non-sporulating rods (0.5×1.0~2.0 μm) with polar multi-trichous flagellation, circular slightly convex smooth shiny yellowish orange colonies on heart infusion agar, oxidative glucose metabolism, positive catalase, oxidase, gelatin liquefaction, citrate utilization, arginine dihydrolase, and fluorescent pigment formation, negative poly-β-hydroxybutyrate accumulation, nitrate reduction, lysine and ornithine decarboxylases, and growth at 28 and 5°C. The strain was inoculated into a medium consisting of starch 2.0%, glycerol 0.5%, Bacto Soytone 1.5%, corn steep liquor 0.5%, NaCl 0.3%, CaCO3 0.3% (pH7.0) and cultured at 23°C for 2 days on a rotary shaker. Azomycin was isolated via adjusting the culture broth to pH 2.0 with HCl, extracting with BuOH, combining extracts, concentrating, further extracting with ethyl ether and 3% aqueous NaHCO3, re-extracting with BuOH at pH2.0, washing, concentrating to an oily residue, triturating with acetone to get a crude powder, then purifying via Sephadex LH-20 column chromatography (developed with MeOH) and silica gel column chromatography (developed with CHCl3-MeOH 20:1), and crystallizing from MeOH to obtain crystals. The antibiotic is an acidic substance, obtained as colorless prisms decomposing above 250°C, soluble in aqueous alcohols and dimethyl sulfoxide, slightly soluble in methanol and ethanol, essentially insoluble in acetone, ethyl acetate, chloroform and water. Elemental analysis and EI-MS (m/z 113, M+) indicated a molecular formula C3H3N3O2 (Anal Calcd: C31.86, H2.67, N37.16; Found: C31.86, H2.92, N37.10). Its UV absorption (λmax EtOH nm (ε): 219 (3,900), 315 (8,600); λmax 0.1N HCl-95% EtOH nm (ε): 219 (3,900), 315 (8,400); λmax 0.1N NaOH-95% EtOH nm (ε): 223 (3,500), 365 (10,700)), IR spectrum, and 1H/13C NMR data (DMSO-d6, TMS) were consistent with azomycin (2-nitroimidazole). Antibacterial spectrum tests against anaerobic bacteria showed it had activity against strains including Peptococcus asaccharolyticus ATCC14963 (MIC 3.13 μg/ml), Peptostreptococcus micros VPI 5464-1 (1.56 μg/ml), Clostridium perfringens ATCC13124 (0.78 μg/ml), Clostridium difficile ATCC17857 (0.78 μg/ml), Bacteroides vulgatus ATCC29327 (0.39 μg/ml), Bacteroides melaninogenicus GAI 0413 (0.39 μg/ml), Fusobacterium necrophorum ATCC25286 (0.78 μg/ml), etc., with MIC values ranging from 0.39 to >100 μg/ml.