Aim To evaluate the bacterial reduction achieved by WaveOne Gold (Dentsply Sirona Endodontics, Ballaigues, Switzerland), Hyflex EDM (Coltene-Whaledent, Altstatten, Switzerland), and XP-endo Shaper (FKG Dentaire SA, La Chaux-de-Fonds, Switzerland) in canals infected with Enterococcus faecalis. The null hypothesis (H-0) was that there would be no difference amongst the NiTi systems in reducing the root canal bacterial load. Methodology Seventy-four mandibular premolar teeth with straight and round root canals were selected. Sixty-two root canals were contaminated with E. faecalis for 4 weeks and subjected to mechanical preparation with one of three single-file NiTi rotary systems (WaveOne Gold, Hyflex EDM, and XP Endo Shaper). Twelve non-contaminated root canals were used as negative controls. Bacterial samples were collected with sterile paper points (Dentsply Sirona Endodontics) before and after rotary instrumentation to quantify the bacterial load. Five roots from each instrumentation group were evaluated topographically from the coronal, middle and apical thirds by SEM. The bacterial reduction was calculated and analysed statistically by the Kruskal-Wallis and Dunn post hoc tests, all at 5% significance.