The prevalence of multidrug-resistant (MDR) pathogens combined with a decline in antibiotic discovery presents a major challenge for health care. To refill the discovery pipeline, we need to find new ways to uncover new chemical entities. Here, we report the global genome mining-guided discovery of new lipopeptide antibiotics tridecaptin A5 and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA5 was explained solely by a Phe to Trp substitution as compared to Oct-TriA1, while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A5. Screening of tridecaptin analogues substituted at position 9 identified Oct-His9 as a potent congener with exceptional efficacy against Pseudomonas aeruginosa and reduced hemolytic and cytotoxic properties. Our work highlights the promise of tridecaptin analogues to combat MDR pathogens.