Objectives (1) To explore the influence of biofilm maturation and timing of exposure on fluoride anticaries efficacy and (2) to explore biofilm recovery post-treatment.

Methods Bovine enamel specimens were utilized in a pH cycling model (28 subgroups [n = 18]). Each subgroup received different treatments [exposure]: sodium fluoride [NaF]; stannous fluoride [SnF2]; amine fluoride [AmF]; and de-ionized water [DIW], at a specific period: early: days 1–4; middle: days 3–6; and late: days 7–10. During non-exposure periods, pH cycling included DIW instead of fluorides. Objective 1: part 1 (cycling for 4, 6, or 10 days). Part 2 (cycling for 10 days). Objective 2: early exposure: three sample collection time points (immediate, 3 days, and 6 days post-treatment); middle exposure: two sample collection time points (immediate, 4 days post-treatment). The enamel and biofilm were analyzed ([surface microhardness; mineral loss; lesion depth]; [lactate dehydrogenase enzyme activity; exopolysaccharide amount; viability]). Data were analyzed using ANOVA (p = 0.05).

Results Objective 1: Early exposure to fluorides produced protective effects against lesion progression in surface microhardness and mineral loss, but not for lesion depth. Objective 2: Early exposure slowed the demineralization process. SnF2 and AmF were superior to NaF in reducing LDH and EPS values, regardless of exposure time. They also prevented biofilm recovery.

Conclusion Earlier exposure to SnF2 and AmF may result in less tolerant biofilm. Early fluoride treatment may produce a protective effect against demineralization. SnF2 and AmF may be the choice to treat older biofilm and prevent biofilm recovery.

Clinical relevance The study provides an understanding of biofilm-fluoride interaction with mature biofilm (e.g., hard-to-reach areas, orthodontic patients) and fluoride’s sustainable effect hours/days after brushing.