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The aim of this study was to investigate the antibacterial activity of 4 endodontic sealers against bacteria planktonic grown or in biofilms commonly detected from persistent and secondary endodontic infections.The antibacterial activity of the sealers AH Plus, TotalFill BC sealer, RoekoSeal, and Guttaflow 2 was investigated for planktonic grown and 24-hour-old biofilms of Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus mutans.AH Plus had high antibacterial activity toward all species investigated, both planktonic and in biofilms. However, the antibacterial activity was lost after 24 hours. TotalFill BC sealer showed marked antibacterial effect on planktonic bacteria up to 7 days after setting. TotalFill BC sealer had lower antibacterial activity against biofilms of S. aureus and E. faecalis compared with AH Plus when direct contact between the sealer and biofilm was investigated and for all species investigated when a membrane was used to separate the biofilm and sealer. Guttaflow 2 and RoekoSeal had no antibacterial activity against planktonic bacteria or bacteria in biofilms.Bacteria in biofilms showed higher susceptibility for AH Plus compared with TotalFill BC sealer during the first 24 hours after setting. Investigating the antibacterial activity of endodontic sealers and materials against bacteria in biofilms is highly important to evaluate the materials' ability to eradicate bacteria from the infected root canal.Freshly mixed AH Plus presented antibacterial properties against all species.TotalFill BC was effective on planktonic bacteria up to 7 days after setting.Guttaflow 2 and RoekoSeal presented no antibacterial activity.