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Caries preventive varnishes containing only fluoride might differ from those containing a combination of fluoride and antimicrobial components in terms of mineralization properties and their impact on the cariogenic biofilm. We compared a fluoride and a fluoride + chlorhexidine (CHX)/cetylpyridinium chloride (CPC) varnish on root caries formation in vitro. One hundred bovine root dentin samples were allocated to five groups (n = 20/group) (1) 7700 ppm fluoride varnish (Fluorprotector S (F)), (2) experimental placebo varnish for F (F-P),