Background: The microorganisms play an important role in pulpal and peri-radicular disease pathogenesis, their eradication by mechanical instrumentation is the ultimate goal of endodontic treatment. Objective: To evaluate the mechanical reduction of Enterococcus faecalis by Rotary ProTaper Gold instrument and Reciprocating Reciproc Blue instrument in apical third of root canal by microbiological culture analysis and scanning electron microscopy. Material and Methods: Sixty mandibular premolars were selected and randomly divided in two groups i.e., Group A (ProTaper Gold) and Group B (Reciproc Blue). The teeth were decoronated at 14 mm from the apex to standardize the length. All the samples were sterilized using autoclave, followed by contamination with Enterococcus faecalis. Contamination was confirmed by SEM evaluation. Pre-instrumentation samples were taken for microbiological evaluation. Instrumentation was done in Group A by ProTaper Gold and in Group B by Reciproc Blue. Post-instrumentation samples were taken for microbiological evaluation. The root canals were then split longitudinally into two halves and apical third of each specimen was examined under SEM. Thus collected data were statistically analyzed using paired ‘t’ test, unpaired ‘t’ test and Mann-Whitney test. Results: Both techniques showed statistically highly significant difference from pre-instrumentation value to post-instrumentation value (p<0.001). The instrumentation in Group B resulted in somewhat better bacterial reduction than in Group A, however no statistically significant difference was found (p>0.05). Statistical analysis by Mann-Whitney test showed no statistically significant difference in mean of SEM scores for bacteria at 1 mm and 3 mm level in both groups (p>0.05). Conclusion: Both instrumentation techniques were effective in reducing bacterial load after mechanical preparation with statistically insignificant difference between them.