Aim <p>This in vitro study aimed to compare the dentinal tubule penetration of a conventional epoxy resin-based sealer (AH Plus) and two bioceramic-based sealers (MTA Fillapex and BioRoot Flow).</p> Methodology <p>Thirty extracted human single-rooted premolars were randomly divided into three groups (<i>n</i> = 10): AH Plus, MTA Fillapex, and BioRoot Flow. Root canals were instrumented using the ProTaper Universal rotary file system. Sealers were labeled with 0.1% Rhodamine Bprior to obturated with the single-cone technique. Roots sectioned at coronal, middle, and apical levels into 1&#xa0;mm thick slices. The sealer penetration area (mm²) and maximum penetration depth (mm) were analyzed using confocal laser scanning microscopy (CLSM). Data were statistically evaluated using one-way ANOVA and LSD post hoc tests (<i>p</i> &lt; 0.05).</p> Result <p>AH Plus demonstrated statistically significantly lower penetration in both penetration area and maximum penetration depth compared to both MTA Fillapex and BioRoot Flow (<i>p</i> &lt; 0.05). Among the bioceramic-based sealers, MTA Fillapex exhibited the highest penetration values in the coronal and middle regions. Furthermore, a pronounced decrease in both penetration area and depth was observed from the coronal to the apical regions in all groups (<i>p</i> &lt; 0.05).</p> Conclusion <p>MTA Fillapex and BioRoot Flow exhibit a superior performance in the coronal and middle regions compared to AH Plus. While the differences in the apical region were less distinct, a consistent decrease in penetration values from coronal to apical was observed across all materials. These findings indicate that material composition and root canal region play critical roles in dentinal tubule penetration.</p>