Antiseptics such as chlorhexidine digluconate (CHX) are widely used in clinical dental practice, but their potential risks, particularly regarding antimicrobial resistance (AMR), are not yet known. This study explores the genomic and transcriptomic mechanisms of CHX adaptation in 3 clinical isolates of Streptococcus spp. and their adapted counterparts. The genomic analysis revealed mutations in genes related to membrane structure, DNA repair, and metabolic processes. Mutations include those in diacylglycerol kinase that occurred in Streptococcus salivarius and the autolysin N-acetylmuramoyl-L-alanine amidase homologues in both Streptococcus mitis and Streptococcus vestibularis, which may contribute to enhanced CHX resistance. Our findings showed stress response genes constantly expressed in all 3 CHX-adapted strains, regardless of acute CHX exposure. Commonly upregulated genes were related to oxidative stress, DNA repair, and metabolic pathway changes, especially amino acid related metabolism. In addition, cell surface restructuring, multiple ABC transporter genes, as well as antimicrobial resistance–associated genes were constitutively expressed. Homologue genes that were significantly upregulated across all 3 species after mutation included recD (DNA repair), potE (amino acid transport), and groEL (stress response). In addition, we saw an increase in a gene associated with the penicillin-binding protein PBP2a in all strains. Beyond these conserved adaptations, we observed species-specific shifts under prolonged CHX exposure. In S. vestibularis, glutathione synthesis genes increased while fatty acid metabolism genes were downregulated. S. salivarius showed elevated expression of genes related to organic anion transport and RNA modification. S. mitis exhibited changes in pyrimidine metabolism, ion homeostasis, and pyruvate dehydrogenase complex genes. Uniquely, S. mitis also showed acute CHX response with upregulation of carbohydrate metabolism and phosphotransferase system genes. These findings highlight the complexity of CHX-induced adaptation, suggesting connections to genetic mutations and emphasizing the need for further research to understand and mitigate AMR risks.