Abstract: Anticipation of root canal bacteria contamination is the foundation of endodontic treatment. The most predominant type is Enterococcus Faecalis strain, along with which several Gram negative enteric rods or Gram positive facultative may also be present. The aim of the present study was to compare the bacterial reduction from the infected root canals taking samples of pre-chemomechanical and post-chemomechanical preparation by ProTaper Universal, WaveOne and NeoNiTi instrumentation systems. Seventy-five systematically healthy patients was selected with permanent mandibular premolar teeth with single root and single canal. They are divided in three: Group I (ProTaper Universal), Group II (WaveOne) and Group III (NeoNiTi), in which each individual pre and post instrumentation sample of microorganism was taken and then analyzed using Paired ‘t’ test, One Way ANOVA with Post HOC comparison using Tukey test for microbiological evaluation. When pre-chemomechanical samples were compared by applying One-Way ANOVA test, the difference in pre-chemomechanical values in all three groups was statistically insignificant (p>0.05). Statistical analysis by applying paired ‘t’ test, One-Way ANOVA and p value showed that there was highly statistically significant change from pre-chemomechanical values to post-chemomechanical values in each study group (p<0.001). There was highly statistically significant difference in CFU count percentage reduction between the pre-instrumentation and post-instrumentation values in all the groups (p<0.001). Group I (ProTaper Universal) showed the highest percentage reduction followed by Group III (NeoNiTi) and Group II (WaveOne). Statistical analysis by paired ‘t’ test and p value showed that the reduction in number of colony forming units from S1 (pre-instrumentation) to S2 (post-instrumentation) was statistically highly significant (p<0.001). It may be concluded that the most effective instrumentation technique in eliminating microorganisms from the root canal was ProTaper Universal system in comparison to WaveOne and NeoNiTi.Abstract: Anticipation of root canal bacteria contamination is the foundation of endodontic treatment. The most predominant type is Enterococcus Faecalis strain, along with which several Gram negative enteric rods or Gram positive facultative may also be present. The aim of the present study was to compare the bacterial reduction from the infected root canal...Show More