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Dental caries is caused by acids from biofilms. pH-sensitive nanoparticle carriers could achieve improved targeted effectiveness. The objectives of this study were to develop novel mesoporous silica nanoparticles carrying nanosilver and chlorhexidine (nMS-nAg-Chx), and investigate the inhibition of biofilms as well as the modulation of biofilm to suppress acidogenic and promote benign species for the first time. nMS-nAg was synthesized via a modified sol-gel method. Carboxylate group functionalized nMS-nAg (COOH-nMS-nAg) was prepared and Chx was added via electrostatic interaction. Minimal inhibitory concentration (MIC), inhibition zone, and growth curves were evaluated. Streptococcus mutans (S. mutans), Streptococcus gordonii (S. gordonii), and Streptococcus sanguinis (S. sanguinis) formed multispecies biofilms. Metabolic activity, biofilm lactic acid, exopolysaccharides (EPS), and TaqMan real-time polymerase chain reaction (RT-PCR) were tested. Biofilm structures and biomass were observed by scanning electron microscopy (SEM) and live/dead bacteria staining. nMS-nAg-Chx possessed pH-responsive properties, where Chx release increased at lower pH. nMS-nAg-Chx showed good biocompatibility. nMS-nAg-Chx exhibited a strong antibacterial function, reducing biofilm metabolic activity and lactic acid as compared to control (p < 0.05, n = 6). Moreso, biofilm biomass was dramatically suppressed in nMS-nAg-Chx groups. In control group, there was an increasing trend of S. mutans proportion in the multispecies biofilm, with S. mutans reaching 89.1% at 72 h. In sharp contrast, in nMS-nAg-Chx group of 25 μg/mL, the ratio of S. mutans dropped to 43.7% and the proportion of S. gordonii and S. sanguinis increased from 19.8% and 10.9 to 69.8% and 56.3%, correspondingly. pH-sensitive nMS-nAg-Chx had potent antibacterial effects and modulated biofilm toward a non-cariogenic tendency, decreasing the cariogenic species nearly halved and increasing the benign species approximately twofold. nMS-nAg-Chx is promising for applications in mouth rinse and endodontic irrigants, and as fillers in resins to prevent caries. [Display omitted] [ABSTRACT FROM AUTHOR]