A new 36-membered ring polyol macrolide antibiotic, dihydroniphimycin, with preventive effect against plant fungal infections, was isolated from the mycelium of Streptomyces hygroscopicus 15. The strain also produces the nigericin complex and antibiotic AN-151 and has been deposited in the Bulgarian National Collection of Industrial Microorganisms and Cell Cultures. This study reports the production, isolation, physicochemical properties, and structure elucidation of dihydroniphimycin (antibiotic AK-15-2). The antibiotic was purified via ethanol extraction of the mycelium, n-butanol partitioning, silica gel column chromatography, and preparative HPLC. The pure compound is a colorless powder with a melting point of 126–128°C, [α]D value of +28.0° (c=0.5, MeOH), soluble in lower alcohols, pyridine, DMSO, and DMF but insoluble in ethyl acetate, chloroform, ether, n-hexane, and water. It reacts positively to 3% vanillin-sulphuric acid, iodine vapor, Dragendorf, and Sakaguchi tests. The UV spectrum in methanol shows absorption maxima at 225sh, 231 (ε=28033), and 241sh nm (indicating a dienic system). The IR spectrum (KBr) exhibits hydroxyl (3391 cm⁻¹), lactone carbonyl (1723 cm⁻¹), and C=C double bond (1646 cm⁻¹) stretching vibrations. High-resolution fast atom bombardment mass spectrometry gave a (M+H)⁺ ion at m/z 1144.5200, corresponding to the molecular formula C59H105N3O18 (calculated 1144.5130). The structure was elucidated using 1D and 2D NMR techniques (¹H-¹H COSY, ¹³C COSY, HMBC), revealing a 36-membered lactone ring, a malonyl residue at C-23, and a monomethylguanidino group. Dihydroniphimycin is most similar to niphimycin A but differs by two mass units (molecular weight 1143 vs. 1141) due to the absence of one double bond.