The effect of an antimicrobial-containing varnish on root demineralisation was investigated. The in situ demineralisation effect on the varnish, with or without the active ingredients chlorhexidine and thymol, was measured by means of microradiography in comparison with no application after a 2-week period in vivo. Furthermore, the effect of one or two applications was investigated by the 10 participants who carried sound intact roots in an appliance for four consecutive 2-week periods. In each period, different roots were mounted in the buccal flanges of a lower prosthesis, the experiment being of a randomized cross-over design. The results show that: (1) A single varnish treatment with active ingredients reduced lesion depth and mineral loss by about 77 and 82%, respectively. (2) The control varnish had no effect, and it can be concluded that antimicrobial-releasing varnishes are promising materials for root caries prevention.