The aim of this study was to estimate the antibacterial and anti-adhesive properties of light-cured pit-and-fissure sealants containing fluoride (Teethmate F-1, TF; UltraSea-1 XT, US) or not containing fluoride (Teethmate A, TA)- and glass ionomer liner/base containing fluoride (Vitrebond, VB) against oral bacteria in vivo. VB inhibited the growth of S. mutans, S. oralis and L. casei aerobically and anaerobically, and TA and US inhibited the growth of only S. mutans aerobically. The numbers of adherent cells mediated by insoluble glucan for all materials were lower than that for the control tooth. The numbers of adhesional cells not mediated by insoluble glucan were larger than that for the control tooth. VB showed the highest concentration of fluoride released throughout the four-week observation period, and had the highest pH value. In this study, VB showed superior antibacterial action and fluoride-release compared with the other three materials.