BACKGROUND: Chelating agents used to remove the inorganic part of the smear layer from prepared canals can alter dentin mineral composition, potentially affecting the adhesion of resin-based root canal cement and sealers. AIM: This study aimed to compare the decalcifying effects of 17% ethylenediaminetetraacetic acid (EDTA), 20% N-acetyl cysteine (NAC), and deionized water (control) on root canal dentin using energy-dispersive X-ray spectroscopy (EDXS). MATERIALS AND METHODS: Sixty-nine extracted human mandibular premolars were randomly divided into three groups, yielding 138 root halves treated with either 17% EDTA, 20% NAC, or a control solution (n = 46). The mineral content was assessed using EDXS, and statistical analysis was conducted with the Kruskal-Wallis test, using an alpha level of 5%. RESULTS: Twenty percent NAC and control groups retained more calcium, magnesium, and phosphorus than 17% EDTA, suggesting better mineral preservation, while 17% EDTA resulted in the lowest mineral content across all elements analyzed. CONCLUSION: Twenty percent NAC exhibited milder decalcifying effects compared to EDTA while preserving mineral content. Twenty percent NAC could be a biocompatible alternative to EDTA for smear layer removal in endodontic treatments.