Four echinomycin antibiotics were isolated from the culture broth of a marine streptomycete, and their structures were determined by a combination of chemical and spectroscopic analyses. Antibiotic activities were measured against drug-resistant and biofilm-forming strains of Staphylococcus aureus and Enterococcus faecalis. Minimum inhibitory concentrations ranging from 0.01 microM to greater than 14 microM clearly defined structure-activity relationships for antibiotic potency. Echinomycin was the most active compound with a MIC of 0.03 microM against methicillin-resistant S. aureus and 0.01 microM against biofilm-forming E. faecalis.