Background <p>Effective root canal disinfection is a critical component of predictable root canal treatment, yet obstacles remain in completely eradicating bacterial biofilms from the entire root canal system. Recent advances in instrument design, such as flat-side instruments, have been proposed to improve root canal disinfection and debridement. While conventional rotary instruments have been thoroughly evaluated, there is a paucity of data on the effectiveness of flat-side multi-file rotary systems in reducing the microbial burden within root canals. Therefore, this exploratory laboratory study was designed to assess the influence of the flat-side design in a full-sequence rotary file system on root canal disinfection compared to its conventional counterpart, aiming to establish initial data to guide further larger-scale research with more holistic microbial evaluations.</p> Methods <p>Thirty mandibular first molars were selected according to specific criteria. After establishing an access cavity and ensuring root canal patency in all samples, the teeth underwent a cycle of autoclave sterilization. <i>Enterococcus faecalis</i> suspension was introduced into the root canals and incubated at 37&#xa0;°C for 21 days. After bacterial contamination, an initial bacterial sampling (S1) was taken, and the specimens were distributed into 2 experimental groups according to the instrument design (<i>n</i> = 10 per group): flat-side full-sequence rotary files group (FS group); and its conventional prototype file system (CP group), along with positive and negative control groups (<i>n</i> = 5). At the end of the root canal preparation, another bacterial sampling (S2) was performed to determine the number of colony-forming units per mL (CFU//mL), and the results were compared with a robust ANOVA and the Games-Howell post hoc test with the significance level set at 5% and a confidence interval of 95%.</p> Results <p>None of the tested endodontic instruments succeeded in completely eradicating the root canal infection. However, the flat-side instruments showed a significant reduction in bacterial load, achieving a mean value of 2.78 ± 0.11, compared to their prototype counterparts, which had a higher mean bacterial load of 3.77 ± 0.32 (p-value &lt; 0.05).</p> Conclusion <p>Instrument design can affect root canal disinfection. Flat-side instruments may be more effective in reducing the bacterial burden from root canals than their traditional counterparts. However, this conclusion was derived from a preliminary investigation conducted under specific conditions. Therefore, to enhance the reliability and generalizability of the current outcomes, it is crucial to conduct further investigations with an adequate sample size across diverse laboratory and clinical settings, as well as variations in root canal morphology, to provide a more comprehensive understanding of the impact of this design on root canal infection.</p> Clinical relevance <p>The present preliminary findings suggest that while complete elimination of infection remains challenging, advancements in instrument design, such as the flat-side instruments, could enhance bacterial reduction within root canals.</p>