Applying a saturated potassium iodide (KI) solution immediately after silver diamine fluoride (SDF) application may affect the inhibitory effects of SDF on biofilm formation. This study compared the efficacy of 38% SDF with and without KI on preventing mixed-species biofilm formation on human root dentin surfaces and assessed ion incorporation into root dentin. The biofilms, composed of , , and , were grown on specimen surfaces treated with either SDF or SDF + KI. After 24 h, the biofilms were evaluated using scanning electron microscopy, live/dead staining, adenosine triphosphate (ATP) assays, colony-forming unit (CFU) counts, and quantitative polymerase chain reaction. A Mann-Whitney test was used to compare the results between the groups. Ion incorporation was assessed using an electron probe microanalyzer. The relative ATP content in the SDF + KI group was significantly higher than that in the SDF group ( < 0.05). However, biofilm morphology and the logarithmic reduction in CFUs and bacterial DNA were comparable across the groups. The SDF + KI treatment resulted in less silver and fluoride ion incorporation than that yielded by SDF alone. The inhibitory effects of SDF and SDF + KI on mixed-species biofilm formation were almost equivalent, although KI application affected the ion incorporation.