Low concentrations (1-10mg/mL) of double antibiotic paste (DAP) have demonstrated antibacterial properties in regenerative endodontics. The aim of this study was to evaluate if DAP made radiopaque (RoDAP) with barium sulfate has antibacterial effects against bacterial isolates from a mature and immature tooth with necrotic pulp. Clinical bacterial isolates were obtained from the canals of mature and immature teeth with necrotic pulps during root canal therapy or a regenerative procedure, respectively. Bacterial isolates were grown anaerobically for three weeks on 4x4mm dentin specimens prepared from extracted human teeth (n=48 per biofilm type). The dentin specimens were allocated into six groups and treated as follows: 1mg/mL RoDAP, 10mg/mL RoDAP, calcium hydroxide (UltraCal), placebo (barium sulfate in methylcellulose), no treatment, and no bacteria or treatment (sterile control). After one week of treatment the biofilm was detached and biofilm disruption assays were conducted to determine the bacterial numbers (CFUs/mL). The data was analyzed using Wilcoxon Rank Sum tests followed by pairwise comparisons. 1 and 10 mg/mL RoDAP as well as calcium hydroxide demonstrated significant antibacterial effects against the tested bacterial isolates. The placebo paste did not demonstrate any significant antibacterial effects. No significant difference in antibacterial effects was found against isolates from both mature and immature teeth regardless of the type of treatment. Both 1 and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from mature and immature teeth with necrotic pulps. RoDAP can be beneficial clinically since its adequate placement within the canal system can be confirmed radiographically.