© International & American Associations for Dental Research 2020. Early childhood caries (ECC) is a chronic disease affecting the oral health of children globally. This disease is multifactorial, but a primary factor is cariogenic microorganisms such as Streptococcus mutans. Biosynthetic gene clusters (BGCs) encode small molecules with diverse biological activities that influence the development of many microbial diseases, including caries. The purpose of this study was to identify BGCs in S. mutans from a high-caries risk study population using whole-genome sequencing and assess their association with ECC. Forty representative S. mutans isolates were selected for genome sequencing from a large-scale epidemiological study of oral microbiology and dental caries in children from a localized Alabama population. A total of 252 BGCs were identified using the antiSMASH BGC-mining tool. Three types of BGCs identified herein—butyrolactone-like, ladderane-like, and butyrolactone-ladderane-like hybrid (BL-BGC)—have not been reported in S. mutans. These 3 BGCs were cross-referenced against public transcriptomics data, and were found to be highly expressed in caries subjects. Furthermore, based on a polymerase chain reaction screening for core BL genes, 93% of children with BL-BGC had ECC. The role of BL-BGC was further investigated by examining cariogenic traits and strain fitness in a deletion mutant using in vitro biofilm models. Deletion of the BL-BGC significantly increased biofilm pH as compared to the parent strain, while other virulence and fitness properties remained unchanged. Intriguingly, BL-BGC containing strains produced more acid, a key cariogenic feature, and less biofilm than the model cariogenic strain S. mutans UA159, suggesting the importance of this BL-BGC in S. mutans–mediated cariogenesity. The structure of any BL-BGC derived metabolites, their functions, and mechanistic connection with acid production remain to be elucidated. Nevertheless, this study is the first to report the clinical significance of a BL-BGC in S. mutans. This study also highlights pangenomic diversity, which is likely to affect phenotype and virulence.