Intertidal red algaeHypnea valentiaeassociatedBacillus amyloliquefaciensMTCC 12716 revealed potential inhibitory effects on the growth of drug-resistant pathogens. In the genome ofB. amyloliquefaciensMTCC 12716, biosynthetic gene clusters encoding antibacterial metabolites were predicted, which might be expressed and contributed to the broad-spectrum anti-infective activity. Three homologue members of the 24-membered macrocyclic lactone family, named as bacvalactones1-3bearing 13-O-ethyl (1); 15-O-furanyl-13-O-isobutyl-7-O-propyl-propanoate (2); and 15-O-furanyl-13-O-isobutyl-7-O-propyl-propanoate-7,24-dimethyl (3) functionalities, were acquired through bioactivity-guided purification. The macrocyclic lactones displayed bactericidal activity against opportunistic pathogens causing nosocomial infections, for instance, methicillin-resistantStaphylococcus aureus(MRSA), vancomycin-resistantEnterococcus faecalis(VREfs), and multidrug-resistant strains ofPseudomonas aeruginosaandKlebsiella pneumoniawith MIC <= 3.0 mu g/mL, whereas standard antibiotics ampicillin and chloramphenicol were active only at concentrations of >= 6.25 mg/mL. The biosynthetic pathway of macrocyclic lactones that are generated by trans-AT polyketide synthases through stepwise extension of an acetyl starter unit by eleven sequential Claisen condensations with malonyl-CoA was established, and the structures were correlated with the gene organization of themlnoperon, which encompasses nine genesmlnA-I (approximately 47 kb in size). The best binding poses for each compounds (1-3) withStaphylococcus aureuspeptide deformylase (SaPDF) unveiled docking scores (>= 9.70 kcal/mol) greater than that of natural peptide deformylase inhibitors, macrolactin N and actinonin (9.14 and 6.96 kcal/mol, respectively), which supported their potential in vitro bioactivities. Thus, the present work demonstrated the potential of macrocyclic lactone for biotechnological and pharmaceutical applications against emerging multidrug-resistant pathogens. Key Points center dot Three antibacterial bacvalactones were identified from the symbiotic bacterium. center dot The symbiotic bacterial genome was explored to identify the biosynthetic gene clusters. center dot Trans-AT pks-assisted mln biosynthetic pathway of the macrocyclic lactone was proposed. center dot In silicomolecular interactions of the bacvalactones with S. aureus PDF were analyzed.