Accumulated evidence has illustrated that secondary caries is the major reason for the failure of amalgam and resin composite restorations. The purpose of this study was to assess the cariostatic effects of aged fluoride-containing restorative materials on the formation of secondary root caries. Fifty sound human molars were selected and randomly assigned to five material groups: non-fluoride-containing amalgam (NA), fluoride-containing amalgam (FA), non-fluoride-containing composite (NC), fluoride-containing composite (FC), and glass-ionomer cement (GIC). After standardized class V cavity preparations and placement of restorations, teeth within each group were randomly divided into two subgroups, "non-aged" and "aged". The aged subgroup was immersed in an inorganic buffer solution for 2 wks before being thermocycled. After being thermocycled and subjected to four cycles of caries formation in a bacterial model system, the teeth were sectioned. Depths of outer lesions and areas of lesions on the cavity walls were measured by polarized light microscopy. The results showed that the FA and GIC groups, whether aged or not, had significantly smaller outer lesion depth than the non-fluoride-containing NA and NC groups. After aging, the FA group demonstrated significantly greater lesion depth (p = 0.0002), while the GIC group exhibited no significant changes in lesion depths. The NA group had a greater wall lesion area than the NC group, while both demonstrated no significant changes following 2 weeks of aging. The FA and GIC groups had similar inhibition areas along the cavity walls, whereas both inhibition areas increased significantly after the aging process. It is concluded that the fluoride-containing amalgam and the glass-ionomer cement, even after a two-week aging process, can still elicit a significant preventive effect on recurrent root caries in an in vitro bacterial model system.