The effect of different medicaments and irrigation solutions on root dentin microhardness (MH) was evaluated depending on time. After initial measurements, MH of 60 maxillary incisors (n = 15 per group) was measured after 3 steps: irrigation with 2.5% sodium hypochlorite, exposure to medicaments [triple antibiotic (TAP) or calcium hydroxide (CH) pastes, 1 week or 1 month)] and final irrigation [20 mL of 17% EDTA or 20 mL 1% phytic acid (PA)]. The percentage of MH reduction was calculated. Data were analysed statistically at P < 0.05. Treatments caused a significant decrease in MH values compared to baseline and pre-measurements (P < 0.05). TAP significantly reduced the MH values of the samples over time (P < 0.05). Removal of TAP after 1 month with PA resulted in a significant reduction in MH compared to removing TAP after 1 week and CH removing after 1 month (P < 0.05). Considering the chemical preparation steps, the most hazardous effect on MH was observed after the first irrigation.