Aim: The aim of this study was to evaluate the smear layer removal and microhardness alteration potential of two different antioxidants as final irrigants on root dentin. Materials and methods: The study aimed to investigate the effects of different irrigants on smear layer removal and microhardness alteration in 90 single-rooted mandibular premolars that were decoronated and instrumented till F2 using ProTaper Gold rotary files with 5.25% sodium hypochlorite (NaOCl) irrigation. The teeth were divided into two groups—the smear layer removal group and the microhardness group. In the smear layer removal group, 45 teeth were randomly assigned to three subgroups based on the final irrigation protocol: group I—ethylenediaminetetraacetic acid (EDTA) (positive control), group II—glutathione, and group III—gallic acid. After the final irrigation, the root canals were thoroughly rinsed with 10 mL of distilled water. Subsequently, the roots were split and observed under a scanning electron microscope (SEM) to assess smear layer removal effectiveness. In the microhardness group, all 45 roots were split into two halves and mounted in acrylic blocks. One-half of each root was used for baseline microhardness measurement, while the other halves were exposed to the experimental irrigants to compare pre and postvalues. The microhardness alteration was then evaluated using a Vickers microhardness tester. Appropriate statistical tests were employed to analyze the data obtained from both the smear layer removal and microhardness tests. Results: In smear layer removal EDTA performed better than glutathione and gallic acid in the coronal third. In the middle third EDTA and glutathione performed equally followed by gallic acid. In the apical third glutathione performed better than EDTA followed by gallic acid. In microhardness alteration analysis, all the tested samples showed alteration in microhardness. Conclusion: Glutathione showed better smear layer removal and less alteration in microhardness and it is as effective in performance as EDTA. Clinical significance: The primary objective of this study was to explore alternative final irrigants to replace EDTA in root canal irrigation. Results obtained from glutathione to gallic acid were found to be effective in terms of smear layer removal and showed comparable results to EDTA. Interestingly, glutathione demonstrated the ability to reverse the adverse effects of NaOCl on root dentin and induced minimal microhardness alteration when compared to the other tested irrigants. These findings suggest that glutathione holds promise as a potential substitute for EDTA in root canal procedures, offering similar smear layer removal capabilities while also exhibiting the added advantage of mitigating NaOCl-induced damage to root dentin.