© 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.In recent years, different biotechnological materials and modalities with antibacterial activity are being developed for oral cavity disinfection. However, the antimicrobial effects of all these materials have not been studied and understood in detail. Thus, the aim of this study was to compare the antibacterial activity of ozone therapy with dentine-bonding agents (containing antibacterial monomer 12-meth-acryloyloxydodecylpyridinium bromide (MDPB) and 10-methacryloyloxydecyl dihydrogen phosphate (MDP) and Ca(OH)2 for deciduous teeth in vitro. The antibacterial effectiveness of the studied materials was determined by using a tooth cavity model on cylindrical cavities created in 90 deciduous second mandibular molars. Streptococcus mutans suspension was inoculated in the cavities. The teeth were distributed into six study groups (five different modalities and a negative control group). Dentine samples, which were collected from the cavities before and after the treatment sessions, were microbiologically evaluated and the materials’ antibacterial activities were compared. There were statistically significiant differences in the S. mutans counts before and after treatment (P < 0.05). In terms of antibacterial efficiency, 60-second O3 treatment was found to be the most successful method, followed by 30-second O3, Clearfil Protect Bond (containing MDPB), Clearfil SE Bond (containing MDP) and Ca(OH)2 treatment. The results from this study suggested that longer exposure to ozone might have more beneficial effects in terms of antibacterial activity for reducing the levels of S.mutans.