Given the favorable antimicrobial properties of zinc oxide (ZnONPs), standard silver (AgNPs), and imidazolium-based silver (Im-AgNPs) nanoparticles, this study aimed to evaluate their influence on the microhardness of root canal dentin. In this experimental study, 40 mandibular premolars were decoronated at the cementoenamel junction and longitudinally sectioned into halves to create 80 specimens. They were randomly allocated to 5 groups (n = 16) and irrigated with ZnONPs, AgNPs, Im-AgNPs, NaOCl, or normal saline (as the negative control) for 15 min. The Vickers Hardness Number (VHN) was measured on each root canal third before and after being soaked in irrigants. Statistical analysis was performed using paired t-test, one-way ANOVA, and post hoc Tukey's test (α = 0.05). Im-AgNPs and ZnONPs irrigants improved the microhardness of root dentin, whereas, AgNPs and NaOCl decreased it. ZnONPs yielded the highest VHN at the coronal third (P˂0.001), while the Im-AgNPs provided the highest VHN at the middle and apical thirds (P˂0.001). The AgNPs group showed the lowest VHN at the apical third. The irrigants containing Im-AgNPs and ZnONPs significantly enhanced the root dentin microhardness. However, the use of AgNPs resulted in decreased microhardness.