Objective: The animal oral microbiota represents a promising reservoir of microorganisms that produce antimicrobial metabolites and compounds capable of modulating bacterial antibiotic sensitivity. Here, we aimed to screen the oral microbiota of alpacas to identify microbial strains exhibiting antimicrobial activity against Gram-negative and Gram-positive bacteria, fungi, and yeasts. Methods: To dissect the underlying antimicrobial mechanisms, we selected Pseudomonas isolates that displayed targeted activity against Gram-negative bacteria and subjected them to metabolomic profiling. Results and Discussion: The analysis revealed that this antimicrobial activity is driven by the production of pyoluteorin, a membrane-active metabolite. Although pyoluteorin alone exhibited limited antimicrobial efficacy, it strongly potentiated the action of clinically relevant antibiotics. Notably, at a sub-cytotoxic concentration of 1.5 μg/mL (IC50 = 2.0 ± 0.3 μg/mL), pyoluteorin sensitized antibiotic-resistant Klebsiella pneumoniae strains, enhancing their susceptibility to the membrane-disrupting antibiotic polymyxin B by an order of magnitude. Conclusions: The future development of bacterio-selective membrane-active agents will facilitate the deployment of such synergistic approaches for the emergency treatment of infectious diseases caused by multidrug-resistant pathogens.