The aim of this study was to compare the antimicrobial performance of the Endox Endodontic System (Lysis Srl, Nova Milanese, Italy), MTAD (Dentsply Tulsa Dental, Tulsa, OK), sodium hypochlorite (NaOCl), and HealOzone (Kavo, Biberach, Germany). Seventy instrumented and initially sterile roots with open accesses and containing a paper point were carried by one volunteer in the oral cavity for 1 week. After removal, samples were taken for microbiological analysis. The root canals were then disinfected with the Endox Endodontic System, MTAD, 3% NaOCl, or HealOzone, and, thereafter, samples were repeated for microbiological analysis. The roots were then sealed and incubated for a further week, after which bacterial growth was again determined. After disinfection, there was a significant decrease in the absolute bacterial count between each disinfection method and the positive control group. There was no statistically significant difference between the NaOCl, MTAD, and HealOzone groups. The Endox device showed the least antibacterial effect with significant differences to MTAD and HealOzone. Bacterial regrowth after 1 week of incubation was detected in all samples of the control group, whereas test groups showed several bacteria-free samples.