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Aim: To evaluate the antibacterial efficacy of double antibiotic paste (DAP), silver nanoparticle (AgNP) gel, and tailored amorphous multiporous bioactive glass (TAMP-BG) in concentrations suitable for regenerative endodontics (RE) against 3-week-old Enterococcus faecalis biofilms after 24 hours and 7 days. Results: Radicular human dentin specimens were prepared and inoculated with E. faecalis to form 3-week-old biofilms. DAP (1 mg/mL), AgNPs 0.02%, and TAMP-BG 100 mg/mL, in methylcellulose gel formulations, were used as antimicrobials. The biofilm disruption assay was done followed by quantification of bacterial colony-forming units and scanning electron microscopic analysis. Results showed that 1 mg/mL of DAP or AgNPs 0.02% provided significant antibiofilm effects at both time intervals. Both DAP and AgNPs significantly reduced bacterial counts and biofilms after 7 days compared with 24 hours. Furthermore, 100 mg/mL of TAMP-BG had a comparable antibiofilm effect, but it was less potent than DAP and AgNPs at both time intervals. Conclusions: DAP (1 mg/mL), 0.02% AgNPs, and TAMP-BG (100 mg/mL) can significantly reduce E. faecalis biofilms. However, complete elimination was only possible with DAP and AgNPs. [ABSTRACT FROM AUTHOR]