The aim of this study was to evaluate the effect of resin infiltration on root caries induced by Streptococcus mutans biofilms. Human premolar specimens were divided to 5 groups: negative control (NC), Clearfil SE Bond (SEB), Icon-etch120s+Icon-infiltrant (HA120), Icon-etch10s+Icon-infiltrant (HA10) and K-etchant10s+Icon-infiltrant (PA10). The resin penetration was observed by fluorescent microscope. Biofilm-induced demineralization was conducted again and observed by swept-source optical coherence tomography and confocal laser scanning microscope. The maximum resin penetration depth (PDmax), lesion depth increase (∆LD), frequency of cervical enamel loss and dentinoenamel junction separation length were measured and statistically analyzed. HA120 showed 138.00±49.25 µm PDmax that was significantly larger than PA10 and SEB (p<0.05). SEB created 136.58±64.73 µm coating layers. HA120 and SEB showed significantly lower ∆LD than NC (p<0.05). Resin infiltration with 120s-HCl pretreatment has got a good penetration ability and preventive effect on root caries, however, an additional risk factor of cervical enamel loss was identified.