BACKGROUND: There are few studies on the influence of methylene blue as a photosensitiser on the mechanical properties and adhesion of glass-fiber posts to intraradicular dentin. Thus, this in vitro study aimed to evaluate the influence of photodynamic therapy with a methylene blue photosensitizer on the Martens hardness, elastic modulus, and bond strength of glass-fiber posts in different thirds of intraradicular dentin. METHODS: Eighty bovine teeth were divided into the following five groups: a control using deionized water, and four other groups according to the methylene blue concentration (50 mg/L or 100 mg/L) and substrate treatment (with or without red laser action). Ultramicrohardness test was used to evaluate the mechanical properties in different regions of the root dentin (n = 8). Push-out analysis was evaluated using a universal testing machine (n = 8). Data were subjected to the Kruskal-Wallis test, followed by Dunn's test for comparing groups, and the Friedman test for comparing thirds (α = 0.05). Representative scanning electron microscopy images were obtained. RESULTS: In general, methylene blue in distinct concentrations, with or without laser action, did not cause differences in the mechanical properties or bond strength in different regions of root dentin (P > 0.05). Methylene blue at a higher concentration, activated with laser, produced lower bond strength values in the middle third of the root canal (P < 0.05). CONCLUSIONS: Methylene blue at 50 mg/L had no influence on the mechanical properties of the bovine tooth and the bond strength of the glass-fiber posts to intraradicular dentin.