This in vitro study measured the effect of calcium hydroxide root filling on the microtensile fracture strength (MTFS) of teeth. A total of 40 extracted human disease-free permanent maxillary incisors were hand and rotary instrumented and vertically compacted with United States Pharmacopeia (USP) calcium hydroxide. The teeth were stored in a moist environment for 7, 28, and 84 days. As a control group, 10 teeth were vertically compacted with gutta percha and sealer. The MTFS of the teeth was measured (Mpa) using an Instron machine. Data were assessed statistically using an unpaired t-test (P value). The intracanal placement of calcium hydroxide weakened the MTFS of teeth by 13.9 Mpa per 77 days: an average of 0.157 MPa day-1. Between 7 and 84 days, the MTFS of the dentin was reduced by 43.9%. This difference was statistically significant (P < 0.05). A statistical difference (P < 0.05) was observed between the mean MTFS of the calcium hydroxide-filled dentin between 7 days (45.7 MPa) and 28 days (35.6 MPa) and also between 7 and 84 days (31.8 MPa). There was also a significant difference (P < 0.05) between the MTFS of the calcium hydroxide-filled dentin after 84 days (31.8 MPa) and the gutta percha-filled dentin (41.3 MPa) when used as a control root filling material. The weakening of the dentin by 23-43.9% following root canal filling with calcium hydroxide provides compelling evidence to re-evaluate the daily usage of this material in endodontic therapy.