Objectives: This study was designed to measure and compare dentin microhardness and MTA (ProRoot, Dentsply Tulsa Dental, Tulsa, OK) dislocation resistance (DR) used in regenerative endodontic treatment (RET) after application of three different intracanal medication(ICM) for 2,4 and 12 weeks’ time intervals. Material and methods: One-hundred sixty eight human maxillary central incisors were selected for the study. Teeth were cut apically 12 mm below and coronally 2 mm above the cemento-enamel junction. Canals were instrumented up to instrument F5 (ProTaper Universal, Dentsply Maillefer, Ballaigues, Switzerland), then Peeso drills (Mani, Tochigi, Japan) were used from No. 1 up to No. 6 , passing 1 mm beyond the apical foramen to obtain larger root canals. Between every 2 consecutive instruments 2 mL of 2.5% sodium hypochlorite (NaOCl) used for canals irrigation , final flush using 5 mL of 2.5% NaOCl and 5 mL of 17% ethylene-diamine-tetra-acetic acid (EDTA, Sigma) followed by 10 mL of distilled water. Samples were randomly divided according to the type of the ICM used inside the canal into 4 equal groups (n = 42); Group 1: Double antibiotic past(DAP) paste: 1:1 mixture of ciprofloxacin (Cipro 500 mg, Schering Plough, Kenilworth, NJ, USA) and metronidazole (Flagyl 500 mg, Sanofi-Aventis, Tours, France), Group 2: Bioactive glass powders (BAG S53P4) of 60 mol% SiO2, 12 mol% P2O5 and 28 mol% CaO composition were prepared through sol gel processing route, all reagents were purchased from Sigma-Aldrich(Dorset, UK). Group 3: Non-setting Calcium hydroxide (CH) (Merk, Germany) was used, and finally, Group 4(Control): where no ICM was applied. Samples were kept in saline solution for either 2, 4, and 12 weeks, randomly selected samples from each group n=14 at each interval where intracanal medication removed, half of them n=7 were subjected to dentin Vickers microhardness test, in remaining half n=7 MTA (ProRoot, Dentsply Tulsa Dental, Tulsa, OK) placed 4 mm deep into the coronal third of the roots having a 4 mm-long chamber, samples were stored for a week at 37 °C at 100 % humidity to allow the complete setting of MTA, then push-out test was used to measure the dislocation resistance DR of MTA. Collected data were analyzed using a two-way ANOVA followed by Bonferroni’s post-hoc test was used for pair-wise comparisons (P ≤ 0.05). Results: showed that both DAP, CH groups of intracanal medication and time interval had a significant decreasing effect on dentine microhardness and MTA dislocation resistance the DR of MTA (P-value <0.001). While, BAG group`s results revealed significant increase in dentine microhardness and MTA dislocation resistance (P-value <0.001). The time factor displayed a significant effect on dentin microhardness and the DR of MTA (P-value <0.001).Conclusion: Regarding situations in this study, intracanal medications type and duration of application used in root canals disinfection through RET must be carefully chosen to avoid negative effect on dentin microhardness or DR of MTA jeopardizing the success of the treatment. BAG (S53P4) showed promising results, further studies needed to complete investigations about it as intracanal medication in RET.