The objective of this study was to evaluate the effects of glycolic acid (GA) (with pH 1.2 and 5) and ethylenediaminetetraacetic acid (EDTA) on the chemical and mechanical properties of dentin to investigate the potential use of GA as final irrigant in the root canal therapy. Specifically, changes in microhardness, smear layer removal, erosion, mineral content distribution, apatite/collagen ratio and flexural strength of mineralized dentin treated with GA were assessed. Saline solution was used as a negative control. Knoop microhardness (KHN) was measured on the root canal lumen of root segments. Dentin beams were used for 3-point flexural strength (σ) test. Scanning electron microscopy (SEM) images of root sections were obtained for evaluation of smear layer removal and dentin erosion on root segments and energy dispersive X-ray spectroscopy (EDS) was used for mineral content distribution. The apatite/collagen ratio (A/C) in dentin powder were examined by Fourier transform infrared (FTIR) spectroscopy. KHN, σ and A/C results were statistically analyzed with ANOVA and Tukey tests (α = 0.05). Smear layer and dentin erosion scores were analyzed with Kruskal-Wallis and Dunn tests (α = 0.05). Root dentin treated with EDTA and GA presented similar KHN regardless of the pH (p > 0.05). However, KHN was significantly reduced in EDTA and GA groups when compared to control group (p<0.001). GA showed the same ability to remove the smear layer and to cause dentin erosion as EDTA. EDS results showed that the GA and EDTA solutions did not alter the dentin mineral content distribution. The apatite/collagen ratio reduced with all irrigant solution and was the lowest with GA pH 5 (p<0.001), while σ was not significantly affected by the experimental solutions (p = 0.559). It can be concluded that GA has similar ability to remove the smear layer than EDTA. GA does not affect negatively the chemical/mechanical properties and it does not increase dentin erosion. The use of GA with low pH seems to promote less change in collagen/apatite ratio, but further studies are needed to establish an ideal clinical protocol. Therefore, this study supports the potential use of GA as an alternative final irrigation solution for root canal preparation.