The aim of this study was to evaluate the effect of glycolic acid (GA) and EDTA on dentin mechanical properties. For the cohesive strength, flexural strength and fracture strength tests, the hourglass of root dentin, dentin sticks and roots standardised to 1 mm thickness were used respectively. ANOVA and Tukey tests were used for statistical analysis (P < 0.05). The results showed that EDTA and GA 17% reduced the cohesive strength values when compared to distilled water (control; P = 0.0022 and P = 0.0016 respectively), whereas the values for GA 10% group were similar to those of the control group (P = 0.093). No statistically significant difference was found among the groups for the flexural strength test (P = 0.1974). Fracture strength test showed that EDTA and GA 17% were statistically similar to each other (P = 0.7694) and statistically inferior to GA 10% (P = 0.0007 and P = 0.0004 respectively). It was concluded that 10% GA showed fewer negative effects on dentin mechanical properties.