The purpose of this study was to determine the effectiveness of EDTA and MTAD at different temperatures as a final irrigant to remove the smear layer after the use of 5.25% NaOCl. Seventy-eight human mandibular premolars with single straight canal were prepared by a crown-down technique using rotary 0.06 taper nickel-titanium files. Final irrigation was performed with EDTA and MTAD at different temperatures. The removal of the smear layer in the coronal, middle and apical level of each canal was examined under scanning electron microscope. No difference was found between the EDTA and MTAD at 4°C, 25°C, and 37°C temperatures regardless of the canal level (coronal, middle and apical) (P = 0.286). In EDTA-25, EDTA-37, MTAD-25, and MTAD-37 groups, the difference among the coronal, middle, and apical levels were statistically no significant (P > 0.05). Our findings showed that EDTA and MTAD at 25°C and 37°C are more effective than EDTA and MTAD at 4°C even in the apical level.