The aim of this study was to evaluate the bacterial reduction promoted by ProTaper Next and Twisted File by comparing to ProTaper Universal and manual technique. Sixty distobuccal root canals of maxillary molars sterilized with ethylene oxide were contaminated with Enterococcus faecalis broth culture. After incubation for 21 days, bacterial samples were collected and cultured on m-Enterococcus agar plates. The root canals were divided into 4 groups, according to the system used for instrumentation: ProTaper Next, Twisted File, ProTaper Universal, and crown down manual technique. Other 8 uncontaminated root canals were control asepsis. Bacterial samples were collected immediately and 7 days after instrumentation. The bacterial reduction was calculated and then made intragroup analysis by paired t-test and intergroup analysis by ANOVA and Tukey tests, all at 5% significance. All techniques significantly reduced the bacterial number in the root canal (p<0.05). ProTaper Next and Twisted File resulted in more bacterial reduction than ProTaper Universal and manual technique (p<0.05). ProTaper Next and Twisted File were similar (p>0.05). It can be concluded that ProTaper Next and Twisted File promote a higher bacterial reduction than Protaper Universal and manual technique.