To compare the effects of different irrigation protocols, with/without laser activation, on the radicular dentine's micro-hardness. Methods: Eighty-two human extracted premolars were decoronated and divided into 7 groups. Roots were longitudinally split into two halves. The micro-hardness was measured for one half before and after irrigation protocols. The groups were; G1: MTAD without laser-activation, G2: MTAD with laser-activation, G3: sodiumhypochlorite (SH) with laser-activation, G4: SH then EDTA with laser-activation, G5: SH then MTAD with laseractivation, G6: SH without laser-activation. G7: distilled water (control). In the two-irrigants groups G4 and G5), samples were irrigated first with SH then with MTAD or EDTA irrigants, which were activated by the laser. The difference between the before-and after-irrigation micro-hardness was calculated to obtain the microhardness difference. Data were analyzed using the Paired Sample-t and Two-ways ANOVA tests at P=0.05. Results: Overall, the mean dentine's micro-hardness after-irrigation (103.1) was lower than before-irrigation (116.1) (P<0.001); except for the distilled-water group, (116.6 and 112.9, respectively) (P=0.075). The microhardness reduction of SH without laser-activation group (32.5) was the greatest (P<0.001). The single-irrigant or laser-activation irrigation protocols caused significantly less micro-hardness reduction compared to the two-irrigants or no laser-activation protocols. The mean micro-hardness reduction of SH and MTAD groups (both with laser-activation) (5.8 and 9.3, respectively) were significantly lower than other groups, but not from that of the control group (3.7). Conclusion:Using irrigants significantly reduced the root-dentine's micro-hardness. Although irrigants agitation by an Er: Yag laser significantly minimized micro-hardness reduction, it did not suppress the adverse effects on dentine micro-hardness when two-irrigants were used.