A new 40-membered ring macrolide antibiotic, malolactomycin A, was isolated from the culture broth of Streptomyces sp. 83-634 (isolated from soil in Nago city, Okinawa Islands, deposited under accession No. FERMP-10771). The strain was cultured at 28°C for 72 hours in a medium containing glucose 2%, soluble starch 1%, meat extract 0.1%, dried yeast 0.4%, soybean flour 2.5%, NaCl 0.2% and K₂HPO₄ 0.005% (pH 7.0). Malolactomycin A was isolated by extracting mycelia with 80% aqueous acetone, followed by butanol extraction, and purified via silica gel column chromatography (eluted with dichloromethane-methanol and butanol-methanol-water mixtures), Sephadex LH-20 chromatography (methanol), and preparative HPLC (Nucleosil 5C18, 82% methanol), yielding 43 mg of white powder. The antibiotic has a decomposition point of 129-131°C, pKa 4.9 in 70% methyl cellosolve, [α]D²⁰ +15.1° (c 0.34, methanol), solubility in lower alcohols, and positive reactions to permanganate, periodate-benzidine, and Sakaguchi tests. HRFAB-MS gave (M+H)⁺ m/z 1230.7890 (calcd. for C₆₃H₁₁₁N₃O₂₀+H⁺ 1230.7833), and elemental analysis (found: C 61.28%, H 9.15%, N 3.37%; calcd.: C 61.49%, H 9.09%, N 3.41%) confirmed the molecular formula. Its structure, a 40-membered macrocycle with a malonate group at C-25 and an N,N'-dimethyl guanidine terminal group, was elucidated using 1D/2D NMR (¹H-¹H COSY, HOHAHA, HMBC, NOE difference) and mass spectral data. In vitro antimicrobial activity showed MIC values of 12.5 µg/ml against Staphylococcus aureus and Bacillus subtilis, >100 µg/ml against Gram-negative bacteria (Escherichia coli, Salmonella typhimurium), 1.6-25 µg/ml against fungi (e.g., Botryotinia fuckeliana, Colletotrichum lagenarium) and 1.6 µg/ml against yeasts (Saccharomyces cerevisiae, Candida albicans). Pot tests demonstrated 97% preventive effect against gray mold (Botritis cinerea) on cucumber at 100 µg/ml, but limited efficacy against anthracnose and brown spot. Acute toxicity (LD₅₀) in mice via intraperitoneal injection was 6.7 mg/kg. Structural comparisons with similar macrolides (e.g., azalomycins, copiamycin, amycin A) revealed differences in ring size, malonyl groups, and methyl residues, confirming malolactomycin A as novel.