Aim: The aim of this study was to investigate the effect of a silver nanoparticle intracanal medicament on root canal dentine microhardness compared with calcium hydroxide. Materials and methods: Silver nanoparticles were prepared and characterised using an optical spectrophotometer and a transmission electron microscope. A total of 60 teeth were selected. Teeth were decoronated and root canals were cleaned and shaped using ProTaper NiTi files. Roots were divided into three groups; calcium hydroxide, silver nanoparticles, as intracanal medicaments, and control group (n = 20). Microhardness was measured using the Vickers test after either 3 days or 1 week (n = 10) in coronal, middle and apical thirds. One-way ANOVA, followed by the Tukey post hoc test, was used to compare between two groups in non-related samples. Results: Results showed that silver nanoparticles showed a significant incremental increase in dentine microhardness (270.50 VHN ± 132.01) than both the control group (155.00 VHN ± 15.11) and the calcium hydroxide group (61.55 VHN ± 11.63), which showed the lowest dentine microhardness during both test periods. Conclusion: The use of silver nanoparticles could provide a substitute to calcium hydroxide as an intracanal medicament that increases the dentine microhardness of root canal-treated teeth. Clinical significance: Nanosilver as an intracanal medication showed a gradual increase of microhardness of dentine over time. Together with its antibacterial effect it could provide a substitute to calcium hydroxide that has a detrimental effect on the dentine microhardness. Keywords: calcium hydroxide, dentine, intracanal medicament, microhardness, silver nanoparticles