To evaluate the preventive effect of Er:YAG laser (EYL) irradiation followed by silver diamine fluoride (SDF) application on dentine with cariogenic biofilm challenge. Twenty-four dentine slices were prepared from extracted sound human third molars. Each slice was cut into four parts for SDF application, followed by EYL irradiation (group SL), SDF application (group S), EYL irradiation (group L) and water (group W). The specimens were subjected to cariogenic biofilm challenge for 12 h, followed by immersion in a buffered remineralising solution containing calcium chloride and sodium hypophosphate for 12 h at 37 °C. Surface morphological changes in the specimens were examined using scanning electronic microscopy. Elemental analysis was performed using energy-dispersive X-ray spectrometry. Micro-mechanical properties were investigated by nano-indentation. The specimen surfaces of groups SL and L showed laser melting contours with narrowed dentinal orifices. Group S showed a partial tubular occlusion. A porous surface was observed in group W, indicating demineralisation. The mean (SD) fluoride weight percentages were 3.93 (0.91), 3.10 (0.61), 0.17 (0.09) and 0.32 (0.07) in groups SL, S, L and W, respectively, (p < 0.001; SL, S > L, W). The mean (SD) micro-hardness values in GPa were 1.84 (0.22), 0.49 (0.13), 0.41 (0.11) and 0.30 (0.06) in groups SL, S, L and W, respectively, (p < 0.001; SL > S > L, W). The mean (SD) elastic moduli in GPa were 75.1 (7.2), 20.0 (1.3), 24.3 (5.2) and 20.2 (2.8) in groups SL, S, L and W, respectively, (p < 0.001; SL > S, L, W). SDF application followed by EYL irradiation on a dentine surface increased its resistance to cariogenic biofilm challenge.