Introduction <p>The present study aimed to evaluate dentinal tubule penetration depth and push-out bond strength of root canal sealers based on conventional zinc oxide - eugenol (ZOE), Nano ZOE, and Cu-doped Nano ZOE.</p> Methods <p>This in-vitro study was conducted on 90 extracted human single-rooted teeth. All samples were prepared using the crown-down technique and irrigated with 5.25% sodium hypochlorite and 17% EDTA. Root canal obturation was performed using the single-cone technique with three sealers: ZOE, Nano ZOE, and Cu-doped Nano ZOE. The specimens were randomly allocated into two equal experimental sets (<i>n</i> = 45 each). The first set was used for evaluation of dentinal tubule penetration depth using Field Emission Scanning Electron Microscopy (FE-SEM), while the second set was used for assessment of push-out bond strength. In each set, samples were equally divided into three groups (<i>n</i> = 15 per group) based on the type of sealer. Data were analyzed using one-way ANOVA, LSD post hoc test, and independent t-test.</p> Results <p>The nano zinc oxide sealer showed the highest tubular penetration depth, while the conventional zinc oxide sealer showed the lowest. Significant differences were observed among the evaluated sealers in tubular penetration depth at the coronal (<i>P</i> &lt; 0.001) and apical (<i>P</i> &lt; 0.001) thirds. In addition, penetration depth decreased significantly from the coronal to the apical thirds in all evaluated sealers. Regarding push-out bond strength, the Cu-doped zinc oxide group was significantly higher than the nano zinc oxide group, and the latter group was significantly higher than the conventional zinc oxide group (<i>P</i> = 0.012).</p> Conclusion <p>Nano zinc oxide sealer demonstrated superior dentinal tubule penetration, while Cu-doped zinc oxide exhibited the highest push-out bond strength, highlighting the potential influence of nanoparticle modification on the performance of zinc oxide–based endodontic sealers.</p>