Background: This study aimed to assess the effects of calcium hydroxide, Biodentine, calcium-enriched mixture (CEM) cement, and mineral trioxide aggregate (MTA) on root dentin flexural strength after a 30-day exposure period. Methods: This in vitro experimental study evaluated 25 freshly extracted sound human incisors with no caries or restorations. The apical 5 mm and the coronal two-thirds of the crowns were cut such that all samples had 10 mm length. Dentin samples (n=20 in each group) were then exposed to 2 mm thickness of calcium hydroxide, Biodentine, CEM cement, MTA, or saline (control) in petri dishes for 30 days. Finally, dentin samples were subjected to a three-point bending test after the intervention, and the flexural strength data were analyzed using one-way ANOVA, Tukey’ s test, and t test. Results: Thirty-day exposure to all four biomaterials decreased the flexural strength of root dentin (P < 0. 05). The four groups were significantly different in terms of the flexural strength of root dentin (P = 0. 001). The flexural strength of root dentin was significantly lower following exposure to calcium hydroxide (P = 0. 003), Biodentine (P = 0. 011), CEM cement (P = 0. 001), and MTA (P = 0. 007) compared to saline. The reduction in strength following exposure to calcium hydroxide was higher than that in Biodentine, CEM cement, and MTA groups (P < 0. 05) while the latter three were not significantly different in this respect (P > 0. 05). Conclusions: In general, all four tested biomaterials decrease the dentin strength although this reduction is more prominent by calcium hydroxide.