Objectives <p>This study aimed to compare the antimicrobial effects of Propolis, Calcium Hydroxide (CH), Triple Antibiotic Paste (TAP), and modified TAP (mTAP) as intracanal medicaments on tubular dentin inoculated with a dual-species biofilm.</p> Materials and methods <p>In this ex vivo study, dentin cylinders were obtained from the root canals of 56 single-rooted extracted teeth, which were instrumented and inoculated with <i>Enterococcus faecalis</i> (<i>E. faecalis</i>) and <i>Actinomyces naeslundii</i> (<i>A. naeslundii</i>) suspensions to allow biofilm formation. After that, the cylinders were randomly assigned to five groups for a 7-day exposure to Propolis, TAP, mTAP (penicillin G), and Calcium Hydroxide (CH), and a no-medicament negative control group. Colonies were counted in dentin samples obtained from 200&#xa0;μm to 400&#xa0;μm depths and analyzed using ANOVA, Shapiro-Wilk, Levene, Tukey, robust Welch, Games-Howell, Fisher’s exact, Wilcoxon, and paired t tests (α = 0.05).</p> Results <p>All medicaments significantly decreased the colony counts (<i>P</i> &lt; 0.05). CH, TAP, and mTAP showed similar optimal efficacy at both depths, whereas Propolis caused a significant reduction in bacterial count only at 200&#xa0;μm (<i>P</i> = 0.047). Effect sizes were very high for both microorganisms. Dentin depth had a significant effect on the bacterial count of both microorganisms, and the load of both microorganisms was significantly lower at 400&#xa0;μm than 200&#xa0;μm depth (<i>P</i> &lt; 0.001).</p> Conclusion <p>TAP and mTAP demonstrated high efficacy, and CH showed acceptable efficacy for elimination of <i>E. faecalis</i> and <i>A. naeslundii</i> dual-species biofilm. Propolis showed lower efficacy, highlighting the need for further modifications to enhance its penetration depth.</p>