Abstract Body (Do not enter title and authors here): Infective endocarditis (IE) is a life-threatening infection of the heart’s inner lining that remains difficult to treat and prevent, especially in individuals with preexisting cardiac conditions. Major etiologic agents include viridans group streptococci such as Streptococcus sanguinis, a beneficial oral commensal, and Streptococcus mutans, the primary cause of dental caries. Despite their contrasting roles in oral health and phylogenetic distance, both species can cause severe IE. Although several IE-associated virulence factors have been identified, a comprehensive and systematic analysis of genes contributing to IE pathogenesis has been lacking. In this study, we conducted a genome-wide screen in S. sanguinis strain SK36 using a vertebrate IE model to evaluate the role of individual genes during infection. Among 2,039 defined open reading frame (ORF) deletion mutants, we identified 146 with significantly reduced in vivo fitness, 83% of which were validated in at least two independent experiments involving 3–5 animals per group. Notably, nearly 95% of these genes had not previously been linked to IE in S. sanguinis or any other species. These genes were enriched in unexpected pathways, including coenzyme A biosynthesis, the shikimate pathway, and rhamnan synthesis, with nearly all components essential for endocardial colonization. To assess the conservation of these findings, we deleted coaC, a coenzyme A biosynthesis gene, in S. mutans strain UA159. The mutant exhibited impaired growth in serum and significantly reduced fitness in the rabbit IE model, mirroring the phenotype observed in S. sanguinis. Collectively, these results uncover previously unrecognized, conserved genetic determinants of streptococcal virulence in IE and identify promising targets for therapeutic intervention and disease prevention.