Vibrio cholerae, the causative agent of cholera, remains a major global health burden despite ongoing initiatives for vaccination and improved sanitation. Much of the V. cholerae lifecycle occurs in aquatic environments, requiring gene functions that enable survival under multiple stresses associated with this niche and during the transition to the human host. Although numerous V. cholerae genome sequences are available, functional annotation across its pan-genome remains incomplete, limiting our understanding of its biology.

In this study, we employed a chemical genomics approach to profile fitness across 104 diverse stress conditions and identify growth phenotypes for unannotated genes using 3,026 single-gene deletion mutants of V. cholerae C6706, a widely used research strain. In total, we identified significant growth phenotypes in 1,658 mutants, of which 285 correspond to currently unannotated genes. Together, these data provide a comprehensive resource for exploring gene function in V. cholerae, supported by detailed quality metrics and open-access tools to facilitate community-driven discovery.