Objective This study aimed to assess the efficacy of Cupral®, a Ca(OH)2 and Cu2+ based materials used in endodontics, against biofilms of the oral species Streptococcus oralis, Streptococcus gordonii and Aggregatibacter actinomycetemcomitans at different maturation stages. Methods Biofilms of the bacterial target species were grown in brain heart infusion (BHI) medium for 1 and 5 days on titanium disks (titanium, grade 4) to collect microbial communities at different stages of biofilm maturation. Biofilms were subjected to different Cupral® concentrations (4-, 15- and 50-fold dilution) to assess the antimicrobial- and biofilm dissolving effect. 0.2% chlorhexidine gluconate (CHX) solution was used as a positive control. Biovolume and antibacterial efficacy were analyzed by live/dead staining in combination with confocal laser scanning microscopy (CLSM) to quantify biofilm detachment and antibacterial efficacy. Results All tested Cupral® concentration showed a strong antibacterial effect on tested bacterial species at all biofilm maturation stages. Efficacy of biofilms detachment was concentration dependent, i.e. higher Cupral® concentrations generally led to increased biofilm detachment. The antibacterial efficacy of tested Cupral® concentration was at least equal to CHX treatment (P=0.03). Conclusion Cupral® shows a strong anti-biofilm efficacy and may be applied for oral biofilm treatment and control in dental disciplines other than endodontics.