Purpose: To evaluate the activity of a methacryloyloxydodecylpyridinium bromide (MDPB)-containing self-etching primer (Clearfil Protect Bond) against Streptococcus mutans and its ability to reduce biofilm formation on standardized experimental Class I restorations in vitro. Materials and Methods: Forty experimental Class I round restorations were prepared on enamel-dentin slabs using different adhesive strategies: group 1 = MDPB-containing adhesive system (Clearfil Protect Bond); group 2 = MDPB-free self-etching adhesive system (Clearfil SE Bond); group 3: MDPB-containing self-etching primer in combination with a fluoride-free bonding agent; group 4: MDPB-free self-etching primer in combination with a fluoride-containing bonding agent; group 5: a three-step etch-and-rinse adhesive system (Adper Scotchbond Multi Purpose). A Streptococcus mutans biofilm was grown for 48 h on the restoration surfaces and subsequently evaluated using scanning electron microscopy on three different areas: enamel, composite, and interface surfaces. Statistical analysis was performed by multiple ANOVA after data transformation. Results: Specimens in groups 2, 4 and 5 showed greater biofilm formation than those in groups 1 and 3 (p 0.001) on all investigated substrates (enamel, composite, and interface areas). Conclusions: Specimens prepared with an MDPB-containing primer exhibited significant decreases in biofilm formation on Class I restorations in vitro. Further in vitro and in vivo studies are required to clarify the role of quaternary ammonium compounds in reducing bacterial biofilm formation on restoration surfaces. Keywords: dentin bonding agents, MDPB monomer, antibacterial agents, biofilms, Streptococcus mutans