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Background: The objective of this study was to compare the efficiency of four commonly used chemicals in their ability to remove smear layer after instrumentation using scanning electron microscope (SEM). Materials and Methods: Seventy-five extracted single canaled teeth of roots ranging 10-12 mm in length were used for the study. Teeth were divided into 4 study groups and 1 control group of 15 teeth each. Standard access to the pulp chambers were performed with diamond burs. The lengths of the teeth were determined by the introduction of a size 15 K-file into the root canal until the tip reached the apical foramen. The working length for preparation of the canal is set 0.5 mm shorter than the measurement. Irrigation was performed using 2 ml of irrigant for every instrument change and finally rinsed using 5 ml of the respective solutions. The roots were then split with a chisel and hammer. One-half of each tooth was selected and prepared for SEM examination. After assembly on coded stubs, the specimens were placed in a vacuum chamber and sputter-coated with a 300 Å gold layer. The specimens were then analyzed using a Philips SEM XL 30. The dentinal wall of the cervical, middle and apical thirds was observed at magnifications of up to ×1000 for the presence/absence of smear layer and visualization of the entrance to dentinal tubules. Photomicrographs (×1000) of these areas on each of the coronal, middle and apical thirds were made Data were analyzed using Kruskal–Wallis test and Mann–Whitney U test. Results: SEM study done on these prepared teeth with the popularly used four chemicals, namely, 3% NaOCl (Group A), 3% NaOCl followed by 17% ethylene diamine-tetra-acetic acid (Group B), 0.2% chlorhexidine (Group C) and 3% NaOCl followed by MTAD (Group D), with distilled water (Group E) which is used as control, revealed that NaOCl showed statistically significant, better cleansing effect than distilled water. Chlorhexidine and NaOCl showed equal kind of efficacy but were statistically significant, with lower efficacy than MTAD. It may be concluded that MTAD appears to be the most effective solution compared to the rest. Conclusion: The study demonstrated that MTAD as a final rinse after the entire instrumentation with 3% NaOCl as irrigant provided the best cleansing in all parts of the root canal system. The smear layer has been shown to hinder the penetration of intracanal disinfectants and sealers into dentinal tubules and has the potential of compromising the seal of the root filling. Degradation of the smear layer after treatment may contribute to leakage and reinfection of the root canal space. Removal of the smear layer reduced the penetration of bacteria through the root canal system after root filling.