We investigated the antibacterial effect of low concentrations of double antibiotic paste (DAP) loaded into a methylcellulose system against bacterial biofilms obtained from mature and immature teeth with necrotic pulps. Standardized radicular dentin specimens were randomly divided into six experimental groups (n = 20). Group 1: 5mg/mL DAP treatment. Group 2: 1mg/mL DAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose. Group 5: No treatment. Group 6: No bacteria or treatment. Clinical bacterial isolates were obtained from mature and immature teeth with necrotic pulps indicated for endodontic regeneration or routine endodontic treatment, respectively. Specimens in each group were inoculated with either bacterial isolates (n = 10) and incubated anaerobically for 3 weeks. Specimens were then treated for one week with the assigned group treatment. Treatments were rinsed with sterile saline and biofilms were detached and spiral plated using biofilm disruption assays. Wilcoxon Rank Sum tests followed by pair-wise comparisons were used for statistical analyses. Treatment of infected dentin with 1 mg/ml of DAP, 5 mg/mL of DAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste. Furthermore, 1 mg/mL of DAP caused complete eradication of biofilm obtained from mature tooth with necrotic pulp. However, the same concentration was not able to completely eradicate biofilm obtained from the immature tooth with necrotic pulp. Low concentrations of DAP (1-5 mg/mL) loaded into a biocompatible methylcellulose system demonstrated significant antibacterial effects against biofilm obtained from both mature and immature teeth with necrotic pulps.