This study aimed to evaluate the fracture resistance of root and sealer penetration after obturation using an epoxy resin sealer AH plus (AH+) and two different bioactive endodontic sealers, i.e., Totalfill BC Hiflow (TF BC), and experimental injectable bioactive glass (Exp.BG). A thermo-sensitive injectable sealer was prepared by using a non-ionic triblock copolymer and bioactive glass. The root canals of human extracted teeth were obturated with the respective sealers. The fracture resistance was analyzed at different time intervals, i.e., days 7, 30, and 90. The morphological and elemental analyses of the fractured roots were conducted with a scanning electron microscopy and a electron dispersive spectroscopy. Sealer penetration depth and the percentage of penetrated sealers into the dentinal tubules were assessed with the confocal laser scanning microscope. Statistical analysis was performed using a one-way ANOVA post hoc Tukey’s test. The mean fracture force in AH+ was significantly higher on day 30 (664.08 ± 138.8 N) compared to day 7 (476.07 ± 173.2 N) and day 90 (493.38 ± 120.18 N). There was no statistically significant difference between the TF BC and Exp.BG at different time intervals. The maximum penetration was observed in the middle region compared to coronal and apical for the Exp.BG, followed by the TF BC and AH+ groups; however, a nonsignificant difference in penetration was found over time. It is concluded that the TF BC group showed overall better fracture resistance than AH+ at day 90. Exp.BG showed comparable sealer penetration to those of TF BC and better than those of AH+.