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The effect of different concentrations of 2-hydroxyethyl methacrylate (HEMA) was evaluated on biofilm formation and preformed biofilm of Streptococcus mitis, Streptococcus mutans and Streptococcus oralis, alone or combined to each other.Twofold serial dilution of HEMA ranged from 12 to 0·75 mmol l−1 was added to Streptococcal broth cultures and mature biofilms in 96-well-microtitre plates to evaluate bacterial biomass and cell viability. HEMA affected the Streptococcal population in a strain-specific way producing few significant effects. A reduction on biofilm formation and a detachment of preformed biofilm was recorded in Strep. mitis ATCC 6249, whereas in mixed cultures, the monomer expressed a general aggregative effect on mature biofilms. A reduction in cell viability was also recorded in an HEMA-concentration-dependent way in each experimental condition studied.These results suggest that the HEMA prevalent effects are both the reduction of bacterial adhesion to a polystyrene surface and the increase in dead cells also characterized by an aggregative status.Understanding the potential effect of HEMA, released from resin-based materials, on oral bacteria may furnish information for surveillance of the risk reduction in secondary caries via hindering biofilm generation.