Background and Aim: Methylene blue and curcumin are effective photosensitizers for inactivation of bacteria. This study assessed the penetration depth of methylene blue and curcumin in presence/absence of smear layer into dentinal tubules. Materials and Methods: Thirty-two human central and lateral incisors were included in this experimental study. The initially prepared specimens were randomly allocated to 4 experimental groups: Group 1: methylene blue with smear layer, group 2: methylene blue without smear layer, group 3: curcumin with smear layer, group 4: curcumin without smear layer. Root specimens were sectioned by a diamond disc at 4 and 8 mm from the apex to obtain apical, middle, and coronal sections. The mean penetration depth was measured at the buccal, mesial, distal and palatal areas on cross sections. ANOVA was used to assess the effect of photosensitizer type, smear layer, and root level on penetration depth. Pairwise comparisons were performed by the Student's t-test. Results: The maximum penetration depth was in the apical third in group 2 (0.98±0.25 mm) and the minimum penetration depth was in the coronal third in group 1 (0.21±0.15 mm); this difference was significant (P=0.001). Smear layer removal from the apical and middle thirds was correlated with higher photosensitizer penetration depth (P=0.000) but this difference was not significant in the coronal third (P=0.6). Curcumin had significantly greater penetration depth in presence of smear layer in all three parts compared with methylene blue (P<0.05). Conclusion:Curcumin can penetrate more into dentinal tubules than methylene blue in presence of smear layer.