This in vitro study evaluated the bacterial reduction provided by the EndoActivator (EA), Easy Clean (EC), passive ultrasonic irrigation (PUI), and XP-Endo Finisher. Eight-four mesial roots of mandibular first molars were instrumented, inoculated with Enterococcus faecalis, and divided into four groups (n. 20). Bacterial reduction in the main canals and dentinal tubules were respectively determined by MTT assays and Live/Dead BackLight technique through confocal laser scanning microscopy (CLSM) at 50, 100, and 150 µm in-depth (n. 10 per group). Statistical analyses were conducted following a significance level of 95% (P < 0.05). A significant statistical difference was just identified between XPF and EC in the main canals. In the dentinal tubules from the main root canals, at 100 and 150 µm in-depths, significant statistical differences were only observed between XPF and EC (P = 0.027) for the former and between XPF and EC (P = 0.011) and XPF and PUI (P = 0.021) for the latter. In the dentinal tubules from the isthmus, at 100 µm in-depth, statistically relevant differences did occur between XPF and EC (P = 0.038) and EC and EA (P = 0.029). At 150 µm in-depth, these differences were only significant by comparing XPF and PUI (P = 0.025) and XPF and EC (P = 0.036). Although no irrigation method could thoroughly disinfect the RCS, bacterial reduction indexes were generally better after using XPF.