This study comprehensively characterized the metabolite profiles of six lettuce varieties and established the correlation between the elucidated profiles and their antivirulence effects. A total of 195 metabolites were annotated using LC-QTOF-MS/MS metabolomics assisted by molecular networking and integrated with chemometrics. Red varieties (red longifolia and lolla rosa) demonstrated higher chlorogenic and chicoric acids suggesting their antioxidant properties. In parallel, amino acids and disaccharides were enriched in romaine longifolia rationalizing its palatable taste and nutritional potential, while crispa, capitata, and lolla bionda presented a high & beta;-carboline alkaloid content. The antibacterial and antihemolytic potential of all varieties against methicillin-sensitive and methicillin-resistant Staphylococcus aureus was assessed and validated by prominent downregulation of & alpha;-hemolysin transcriptional levels in both strains. Moreover, correlation analysis revealed sesquiterpenes, & beta;-carboline alkaloids, amino acids, and oxy-fatty acids as the main bioactives. Results emphasize lettuce significance as a functional food and nutraceutical source, and highlight varieties naturally rich in antibacterial agents to adapt breeding programs.