OBJECTIVE The aim of this study was to investigate the caries prevention efficacy of chlorhexidine-thymol (CHX-T) varnish on newly erupted permanent first molars. METHODS Fifty-seven six- to eight-year-old school children were included in a program of sequential CHX-T varnish application. For inclusion, they had to have at least two homologous, newly erupted first permanent molars with visually sound occlusal surfaces. A clinical examination was used to determine the molar eruption stage, biofilm presence, and whole caries status. There were 99 pairs of molars in the study population. A split-mouth design was used where each child randomly had one first molar treated with six applications of CHX-T varnish, and the other with a placebo varnish, every 15 days for 75 days. The children were then revaluated for caries one year following the conclusion of treatment. RESULTS Fourteen pairs of teeth presented incipient enamel caries lesions in both molars (one CHX-T and one placebo varnish), four developed lesions in the placebo-treated molars only, and eight developed lesions in CHX-T varnish-treated molar only. No significant statistical differences were found between the two groups with regard to caries increment (p = 0.20). CONCLUSIONS Six applications of CHX-T varnish had no protective effect against caries development.