Purpose: To evaluate fluoride release and biofilm formation on resin-based composites (RBCs) including surface pre-reacted glass ionomer (S-PRG) filler particles. Materials and Methods: Specimens were prepared from experimental RBCs including different fractions of S-PRG fillers (0/10/30/50/70% w/v). RBCs were light cured against mylar strips (MYL), and 50% of the specimens were additionally polished to a high gloss (POL). Surface roughness (SR), surface free energy (SFE) and fluoride release were determined. Streptococcus mutans biofilm formation (SMBF) was simulated for 48 h and 120 h; adherent viable biomass was assessed using an MTT-based assay. Results: The highest SR was identified for POL specimens manufactured from the RBC with a filler fraction of 70%. For all specimens and surface treatments, polishing caused an increase in surface free energy. For both MYL and POL specimens, increasing the filler fraction coincided with an increased release of fluoride; a higher release of fluoride was identified for POL specimens with filler fractions of 50% and 70% in comparison to their MYL counterparts. Release of fluoride was lower after 120 h than after 48 h. No differences in SMBF were identified between MYL and POL specimens with identical filler fractions after 48 h of biofilm formation; with increasing filler fractions, a tendency towards decreasing SMBF was observed. After 120 h, less SMBF was identified for POL specimens with filler fractions of 30%, 50% and 70% in comparison to corresponding MYL specimens. Conclusion: The inclusion of S-PRG fillers and an effective surface treatment may reduce biofilm formation on RBCs. Keywords: biofilm, Streptococcus mutans, resin-based composite, AFM, surface free energy, fluoride