Objective. This study aimed to test the hypothesis that aggressive dentin removal through greater-tapered instrumentation reduces the intracanal bacteria more effectively than conservative dimension instrumentation. Material and methods. Twenty extracted human lower premolar teeth were used. After extirpation of the pulps, the teeth were autoclaved and immersed in a broth inoculated with Enterococcus faecalis and incubated for 7 days to allow infection of the dentinal tubules. The teeth were divided into 2 experimental groups, each comprising 10 teeth. The teeth were instrumented either with ProTaper or with Hero Shaper nickel-titanium rotary instrumentation techniques. It was calculated that ProTaper theoretically has the potential to remove at least twice the dentin volume compared with Hero Shaper. The apical preparation was standardized to file size 30. Saline solution was used for irrigation. Bacteriological samples were taken before and after instrumentation and plated onto tryptic soy agar, and the reduction in numbers was calculated. Results. Both instrumentation techniques significantly reduced the number of bacteria in the root canal (p < 0.05). Reduction in absolute bacterial numbers was up to 98%. There was no statistically significant difference between the two techniques. Conclusions. Preparation with an instrumentation technique removing substantial amounts of dentin did not reduce the intracanal bacteria more effectively than a more conservative instrumentation technique.