Aim: To assess impact of two novel chelating agents; Silver-Citrate root canal irrigation solution (BioAkt), apple cider vinegar (ACV) in comparison to ethylenediaminetetraacetic acid (EDTA) on the microhardness of root canal dentin. Methodology: Forty single-rooted teeth were gathered, decoronated, root canals were instrumented and roots were divided longitudinally. According to the chelating agent, the specimens were split evenly among four groups; Group 1: BioAkt, Group 2: ACV, Group 3: EDTA, and Group 4: distilled water as a control. At all root levels, the dentin microhardness was assessed both before and after chelating agents were applied. The percentage of microhardness reduction were calculated and the collected data were compared using Two-way Analysis of Variance (ANOVA) and Tukey's tests (P ≤ 0.05). Results: At all root levels, the microhardness of the radicular dentin was significantly reduced by all irrigating solutions as compared to the pre-immersion values (p≤0.05) except for group 4. 17% EDTA showed the highest percentage of microhardness reduction followed by BioAKT, ACV and the least values were recorded for distilled water. Group 3 was significantly different than group 2 and group 4 (control) was significantly different than all tested groups at all root canal levels (P ≤ 0.05). Difference of percentage of microhardness reduction among root levels in all groups were insignificant (p>0.05).Conclusions All tested final irrigating solutions have negative effect on dentin microhardness. Both BioAKT and ACV can be used as safer alternatives to EDTA regarding their effect on microhardness of dentin