We investigated the direct and indirect (residual) antibacterial effects of various concentrations of triple antibiotic paste (TAP) loaded into a methylcellulose system. Enterococcus faecalis (E. faecalis) was grown on sterilized dentin blocks (n = 60) and treated with clinically used TAP (1,000 mg/mL), low concentrations of methylcellulose-based TAP (100, 10, and 1 mg/mL), placebo paste, or 1.5% NaOCl (n = 10). The pastes were then removed, and biofilm disruption assays were performed. Additional dentin blocks (n = 120) were pretreated with the same experimental groups (n = 20). The pastes were rinsed off, and the samples were immersed independently in phosphate-buffered saline for 2 and 4 weeks (n = 10). E.faecalis was then grown on the dentin blocks, and biofilm disruption assays were performed. Fisher's Exact and Wilcoxon rank sum tests were used for statistical analyses. With regard to direct antibacterial effects, all treatment groups demonstrated complete eradication of biofilms in comparison to placebo paste, while 10 mg/mL of TAP or higher provided substantial residual antibacterial effects. However, dentin treated with 1 mg/mL of TAP or 1.5% NaOCl did not provide substantial residual antibacterial effects. Dentin pretreated with 10 mg/mL of TAP or higher exhibited extended residual antibacterial effects and can thus be used during endodontic regeneration.(J Oral Sci 58, 575-582, 2016).