Compounds that specifically target pathogenic bacteria are greatly needed, and identifying the method by which they act would provide new avenues of treatment. Herein we report the concise, high-yielding total synthesis (eight steps, 35% yield) of promysalin, a natural product that displays antivirulence phenotypes against pathogenic bacteria. Guided by bioinformatics, four diastereomers were synthesized, and the relative and absolute stereochemistries were confirmed by spectral and biological analysis. Finally, we show for the first time that promysalin displays two antivirulence phenotypes: the dispersion of mature biofilms and the inhibition of pyoverdine production, hinting at a unique pathogenic-specific mechanism of action.