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Abstract: Objectives: Since secondary caries is one of the main reasons for replacing restorations, this study assessed the effects of different restorative materials on the microbiological composition of dental biofilm and on enamel demineralisation around the restoration. Methods: A randomized, double-blind, split-mouth in situ design was conducted in one phase of 14 days, during which, 20 volunteers wore palatal devices containing five human dental enamel slabs. Each slab was randomly restored with one of the following materials: Filtek-Z-250/Single Bond, control group (composite resin), Permite (amalgam), Fuji II (encapsulated resin-modified glass ionomer), Vitremer (resin-modified glass ionomer) and Ketac Molar (conventional glass ionomer). The volunteers used fluoride dentifrice, 3×/day and a 20% sucrose solution was dripped onto the slabs 8×/day. The biofilm formed on the slabs was analyzed to determine the counts of total streptococci, mutans streptococci and lactobacilli. Enamel demineralisation was determined by cross-sectional microhardness (CSMH) at 20 and 70μm from the margin of the restoration. Kruskal–Wallis and analysis of variance, followed by least mean squares (LMS) test, were used to evaluate microbiota and CSMH among the groups. The significance level used was 5%. Results: No statistically significant differences were found in the cariogenic microbiota grown on the slabs. At a 20-μm distance, only Fuji II statistically differed from the other groups, showing the lowest demineralisation. At 70μm, Fuji II significantly inhibited demineralisation when compared to Permite, Filtek-Z-250 and Ketac Molar. Conclusions: In the context of fluoride dentifrice and under the cariogenic exposure conditions of this study, only the encapsulated resin-modified glass ionomer material provided additional protection against secondary caries. [Copyright &y& Elsevier]