Application of intracanal medicaments may affect the physical properties of root dentine. Calcium hydroxide (CH), a gold standard intracanal medicament, has proven to decrease root dentine microhardness. A natural extract, propolis, has been shown to be superior to CH in eradicating endodontic microbes, but its effect on the microhardness of root dentine is still not known. This investigation aims to evaluate the effect of propolis on root dentine microhardness compared to calcium hydroxide. Ninety root discs were randomly divided into three groups and treated with CH, propolis, and a control. A Vickers hardness indentation machine with a load of 200 g and dwell time of 15 s at 24 h, 3, and 7 days was used for microhardness testing. ANOVA and Tukey’s post hoc test were used for statistical analysis. A progressive decrease in microhardness values was observed in CH (p < 0.01), whereas a progressive increase was observed in the propolis group (p < 0.01). At 7 days, propolis demonstrated the highest microhardness value (64.43 ± 1.69), whereas CH demonstrated the lowest value (48.46 ± 1.60). The root dentine microhardness increased over time when propolis was applied, while it decreased over time after application of CH on root dentine sections.