Aim and objective: The aim and objective of this study was to investigate the contact time effect of 1 minute and 3 minutes of 0.5% chitosan nanoparticle as a final irrigation solution on microhardness and surface roughness of root canal dentin. Materials and methods: This study was divided into two experiments, namely microhardness and surface roughness. Both experiments used 56 mandibular premolars, which assigned to 28 teeth of each study. Twenty-eight teeth were then divided randomly into two groups of 14 teeth. Group I was final irrigated using 17% ethylenediaminetetraacetic acid (EDTA) and group II with 0.5% chitosan nanoparticle. Each group was further assigned into two groups of seven each according to the contact time, group I, 1-minute and group II, 3-minute contact time. The microhardness test was carried out using the Vickers microhardness tester while the surface hardness test using the surface roughness measuring instrument. The data from each study were statically analyzed using two-way ANOVA followed by the least significant difference (LSD) test with a significance level of 95%. Results: Contact time of 1 minute using 0.5% chitosan nanoparticles produced the greatest microhardness and lowest surface roughness of root canal dentin, while contact time of 3 minutes using 17% EDTA had the lowest microhardness and highest surface roughness (p < 0.05). Conclusion: The contact time of 1 minute using 0.5% chitosan nanoparticle caused the least effect on the microhardness and surface roughness. Clinical significance: The findings of this study revealed that contact time of 1 minute using 0.5% chitosan nanoparticle caused the least effect on both microhardness and surface roughness. Thus, the contact time of 1 minute is suggested to be employed when using 0.5% chitosan nanoparticle for the final irrigation solution in the clinic.