Saccharomicins A and B, two new heptadecaglycoside antibiotics, were isolated from the fermentation broth of the rare actinomycete Saccharothrix espanaensis. They represent a novel class of bactericidal antibiotics that are active both in vitro and in vivo against bacteria and yeast (MICs: Staphylococcus aureus, <0.12 to 0.5; vancomycin-resistant enterococci, 0.25 to 16; gram-negative bacteria, 0.25 to >128; and yeast, >128 μg/ml), including multiply resistant strains. Saccharomicins protected mice from lethal challenges by staphylococci (subcutaneous 50% effective dose range of 0.06 to 2.6 mg/kg of body weight, depending on the S. aureus strain). The 50% lethal dose by the subcutaneous route was 16 mg/kg. Mechanistic studies with Escherichia coli imp and Bacillus subtilis suggested complete, nonspecific inhibition of DNA, RNA, and protein biosynthesis within 10 min of drug treatment. Microscopic examination of drug-treated cells also suggested cell lysis. These data are consistent with a strong membrane-disruptive activity. The antibacterial activities of the saccharomicins against gram-positive bacteria were unaffected by the presence of Ca2+ or Mg2+, but activity against gram-negative bacteria was substantially reduced.