OBJECTIVE To evaluate the effect of ionizing radiation and cariogenic biofilm challenge using two continuous flow models, normal and reduced salivary flow, on the development of initial root-dentin caries lesions. METHODS Microcosm biofilms were grown under two salivary flow rates (0.06 and 0.03 mL min-1) and exposed to 5% sucrose (3 × daily, 0.25 mL min-1, 6 min) dripped over non-irradiated and irradiated root-dentin blocks for up to 7 days. The vibration modes of root dentin, matrix/mineral (M/M), and carbonate/mineral (C/M) ratios were evaluated by FTIR. The mineral density was assessed by micro-CT. RESULTS With normal salivary flow, FTIR revealed an increase in the organic matrix (amide III) and a decrease in the mineral phase (ν4, ν2 PO43-, AII + ν2 CO32-, C/M) in caries lesions. Irradiated dentin exhibited a reduction in the mineral phase (ν1, ν3 PO43-, ν2 CO32-, C/M). Differences in mineral densities were not significant. With reduced salivary flow, FTIR also revealed increased organic matrix (amide III) for irradiated caries lesions and decrease in mineral phase (v4, v2 PO43-, v2 CO32-, and C/M) in caries lesions. ν1, ν3 PO43- precipitated on the surface of irradiated dentin and a lower mineral density was observed. CONCLUSIONS Initial caries lesions differed between non-irradiated and irradiated dentin and between normal and reduced salivary flow rates. Significant mineral loss with exposure of the organic matrix and low mineral density were observed for irradiated dentin with a reduced salivary flow rate. CONCLUSIONS Ionizing radiation associated with a reduced salivary flow rate enhanced the progression of root-dentin caries.