BACKGROUND : This study aimed to assess the effect of ultrasonic activation of photosensitizer on the efficacy of Antimicrobial Photodynamic Therapy (aPDT) against Enterococcus faecalis and penetration depth of photosensitizer. METHODS : In this ex vivo study, mature microbial biofilm of E. faecalis was formed in the root canals of 58 single-rooted single-canal mandibular incisors following their decoronation. The roots were longitudinally sectioned by a diamond disc and split into halves by a chisel. The E. faecalis biofilm was quantified and the penetration depth of photosensitizer was determined by the microbial viability assay and stereomicroscopic analysis in the following three study groups: (1) Ultrasonically activated 5.25% sodium hypochlorite (NaOCl) for 20 s, (2) aPDT using methylene blue (MB) plus 660 nm diode laser with 150 mW power for 1 minute, and (3) ultrasonically activated MB for 20 s followed by aPDT as in group 2. Independent sample t test and one way ANOVA were used to compare the dye penetration depth and microbial load, respectively in the apical and coronal regions among the groups. RESULTS : The penetration depth of photosensitizer in group 3 was significantly greater than that in group 2 (P < 0.05). The E. faecalis count in all three experimental groups was significantly lower than that in the control group (P < 0.05). Groups 1 and 3 were significantly superior to group 2 in terms of reduction in microbial count but the difference between groups 1 and 3 was not significant (P > 0.05). CONCLUSIONS : Ultrasonic activation of photosensitizer in aPDT increases the penetration depth of photosensitizer into the dentinal tubules and enhances its antibacterial activity. UNASSIGNED : Ultrasonic activation of photosensitizer in aPDT enhances its penetration depth into dentinal tubules and increases antibacterial efficacy. There was no significant difference between antibacterial effects of aPDT + ultrasonic and ultrasonic activated NaOCl.