This study aimed to investigate the influence of immersion duration and the type of immersion solution on the outcome of push-out bond strength (POBS) tests. Root canals of 120 straight single-rooted teeth were instrumented to a diameter of 1.5 mm and irrigated with 5 mL of 3% NaOCl. Four horizontal slices with a thickness of 1 mm were cut, representing the mid-portion of the root. The specimens (n = 480) were irrigated with 17% ethylenediaminetetraacetic acid(EDTA) for 60 seconds, then twice with distilled water (DW) for 30 s each. The canals were filled with either AH Plus (Dentsply Sirona, Konstanz, Germany) or BioRoot RCS (Septodont, St. Maur-des-Fossés, France) (n = 240). Separated into four groups per type of sealer (n = 60), the specimens were incubated at 37 °C covered with gauze moistened in DW or phosphate-buffered saline (PBS) for either one or eight weeks. Dislodgement resistance was measured and POBS was calculated. Statistical analysis was performed using the analysis of variance (ANOVA) test and the Student–Newman–Keuls test (p = 0.05). AH Plus showed higher POBS when stored in PBS compared to DW, irrespective of the incubation period (p < 0.05). BioRoot RCS displayed higher POBS when stored in DW compared to PBS after eight weeks of incubation (p < 0.05). No difference was found after one week of incubation (p > 0.05). Irrespective of the sealer or the immersion solution, POBS decreased from one week to eight weeks (p < 0.05). Mixed failure modes were found in all groups irrespective of sealer, immersion medium, or immersion period. POBS decreased after a longer incubation time in both immersion solutions. Duration of immersion and the type of immersion solution had a significant impact on the outcome of the POBS testing protocol.