Introduction A smear layer forms on the root canal walls as the consequence of root instrumentation. The smear layer formed in such a way considerably influences the quality of root obturation and endodontic treatment outcome. Objective The aim of this study was to ultrastructurally analyze the surface of intracanal dentine after removal of the smear layer by the solution of doxycycline, citric acid and detergent Tween-80 (MTAD). Methods The study involved 60 single-rooted, extracted, human teeth divided into four groups. All samples were instrumented by a step-back technique and manual K-files, and rinsed during instrumentation by 2% CHX and H2O2. The first group of samples was exclusively rinsed by CHX and H2O2; in the second group, besides using CHX and H2O2, MTAD solution was used for the final irrigation. The samples which were rinsed by distilled water (+ control) and the samples rinsed by 5.25% NaOCl and 17% Na EDTA (-control) served as control groups. All samples were observed under the scanning electronic microscope JEOL-JSM-5300. The coronary, middle and apex thirds of the radix region were analyzed. Results The obtained results of the SEM analysis showed that the application of 2% chlorhexidine and hydrogen peroxide did not give clear dentine walls, and the smear layer could not be removed completely. The application of the same combination (CHX and H2O2), added by the final MTAD irrigation solution, led to the efficient removal of the smear layer, while the morphological structure of dentine surface remained unchanged. Statistical analysis showed that canal walls in the experimental group with MTAD as the final irrigation were significantly clearer compared to the control group (p<0.001). Conclusion Based on the obtained results, it can be concluded that MTAD is an efficient solution for the final irrigation of the canal system.