The antimicrobial and anticariogenic properties of a copper varnish (experimental mixture of Doc's Best Red Copper cement and Copalite varnish, Cooley and Cooley, Ltd, Houston, TX, USA: designated in this study as "Copper Seal") on the root surface were evaluated in an in vitro microbial caries model. Fifty-six human root specimens were prepared from anterior teeth and randomly divided into four groups: Groups 1 and 3-Copper Seal; Group 2-chlorhexidine varnish, the positive control (Cervitec, Ivolcar Vivadent, Schaan, Liechtenstein) and Group 4-a negative control that received no treatment. The varnishes were painted in Groups 1, 2 and 3, then visually removed after 24 hours in Group 1. The specimens were demineralized in a microbial caries model for five days. Plaque was collected from the specimens to obtain bacterial colonization numbers, then the specimens were sectioned and analyzed for lesion extent using Confocal Laser Scanning microscopy. There were no significant differences (p>0.05) among the four groups in terms of bacterial count. Regarding caries lesion development, the group with copper varnish visually removed (Group 1) and the non-treated group (Group 4) had significantly greater total area caries lesions and total lesion fluorescence than the copper varnish without removal group (Group 3) and the chlorhexidine group (Group 2). Therefore, it was concluded that copper and chlorhexidine varnishes have anticariogenic effects on root surfaces, as tested in this model.