ObjectivesTo evaluate the effect of experimental solutions containing TiF4/NaF and chitosan on bacterial species and on enamel caries prevention. MethodsMicrocosm biofilm was produced from human saliva mixed with McBain saliva (0.2% sucrose) on bovine enamel for five days, under 5% CO2 and 37 °C. From the second day until the end, the treatments were applied (1 × 60 s/day): (1) NaF (500 ppm F−, positive control); (2) TiF4 and NaF (TiF4: 190 ppm Ti4+ and 300 ppm F−; NaF: 190 ppm F−); (3) similar to 2 plus 0.5% chitosan (Ch 500 mPas, 75% deacetylation); (4) phosphate buffer solution (negative control); and (5) 0.5% chitosan (Ch 500 mPas, 75% deacetylation). CFU counting was performed for total microorganism, total streptococcus, total lactobacillus and Streptococcus mutans. Enamel demineralization was measured by transverse microradiography-TMR. The data were compared using ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p < .050). ResultsNo differences were found between the treatments with respect to CFU counting (ANOVA, p > .050). Enamel treated with TiF4/NaF plus chitosan solution presented the lowest demineralization compared to the negative control and pure chitosan solution. On the other hand, this experimental solution did not significantly differ from TiF4/NaF and NaF solutions, being all of them able to significantly reduce mineral loss (50–74%), but only TiF4/NaF plus chitosan reduced lesion depth (55%) compared to the negative control (p = .001). ConclusionTiF4/NaF plus chitosan solution had no antimicrobial effect, but it was able to reduce enamel caries development in 79% compared to control under this model. Clinical significanceThis study showed that TiF4/NaF plus chitosan solution had no antimicrobial effect, but it was able to reduce enamel caries development under a microcosm biofilm model.