Aim The aim of this in vitro study was to compare the efficacy of root canal wall debridement following hand versus LightSpeed(TM) instrumentation. Methodology Twenty recently extracted single-rooted teeth were paired and randomly placed into two treatment groups of 10 teeth each. In group 1, a step-rooted instrumentation without: initial coronal flaring with stainless steel Hedstroem fries was used; group 2 was instrumented with Ni-Ti LightSpeed(TM) instruments, Both groups had the same irrigation regimen: 2.5% NaOCl and a 15% EDTA solution, The teeth were then decoronated and each root split longitudinally inter two halves to be examined using the scanning; electron microscope (SEM). The presence of superficial debris and smear layer was evaluated by a standardized grading system, and the resulting scores submitted to nonparametric statistics. Results Under the conditions of this study, the removal of superficial debris was generally excellent with both canal preparation. techniques resulted in variable presence of residual smear layer, with a canal wall covered by smear layer as the predominant characteristic. Generally, the amount of smear layer was greater in the apical than in the middle third of the root, however, this difference was statistically significant (P < 0.005) only in hand-instrumented teeth. The use of LightSpeed(TM) instruments was associated with significantly more (P < 0.05) smear layer presence in the middle region of the root when compared with hand instrumentation. In addition, less smear layer was present in the apical region following LightSpeed(TM) instrumentation than stainless steel hand files, but this difference was not statistically significant. Differences in debridement between the two halves of the same root were more evident with LightSpeed(TM) than manual instrumentation, however, there was no statistical significance. Conclusions It may be inferred that the choice between hand and LightSpeed(TM) instrumentation should be based on factors other than the amount of root canal debridement, which does not vary significantly according to the instruments used.