In this paper, we wish to report the isolation and structural elucidation of Fragin, a new plant growth inhibitor produced by Pseudomonas fragi, a soil bacterium selected using a chlorella-based screening method. The bacterium was cultured in a medium containing 0.02% K2HPO4, 0.05% peptone, 0.02% yeast extract, and 1% glucose. Fragin was isolated via benzene extraction (after adjusting the broth to pH 5 with dilute sulfuric acid), washing with 5% sodium bicarbonate and extraction with 5% sodium carbonate, acidification back to pH 5, re-extraction with benzene, crystallization from n-hexane, cupric acetate precipitation (to form a blue-violet salt), treatment with hydrogen sulfide to remove copper, and final recrystallization from n-hexane, yielding pure colorless plates with m.p. 80°C and [α]D -122°. Elemental analysis and molecular weight determinations gave the molecular formula C13H27O3N3. Spectroscopic (UV, IR, NMR) and chemical studies (spontaneous denitrosation to a dimer, hydrolysis to oxime and ketone, caprylic acid production from hydrolysis) revealed the presence of an N-nitrosohydroxylamino group and a secondary amide linkage. The structure was confirmed by the synthesis of racemic Fragin. Biologically, Fragin inhibited chlorella growth at 20 ppm, lettuce seedling root growth at 10 ppm and hypocotyl expansion at 25 ppm, and Aspergillus niger growth at 20 ppm.