Purpose <p>Investigating dentin strengthening effect of an aqueous extract of Nigella sativa seed (AEN), Er,Cr:YSGG laser (L), or their combination (AENL).</p> Methods <p>Chemical analysis of AEN was carried out using gas chromatography-mass spectrometry (GC–MS) and inductively coupled plasma mass spectrometry (ICP-MS). Twenty extracted human posterior teeth were cut into 40 dentin samples. Samples were randomly distributed into 4 groups (<i>n</i> = 10) according to the prospective surface treatment (0.5 W power Er,Cr:YSGG Laser (L), AEN paste (AEN), AEN paste followed by laser (AENL), and light-cure calcium hydroxide (CH)). Dentin samples were artificially demineralized before surface treatments. Vickers microhardness was evaluated before demineralization (T0), after demineralization (T1), and after surface treatment (T2). Dentin samples were examined using scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX). Data analysis of microhardness means and standard deviations was performed using repeated measure ANOVA and one-way ANOVA.</p> Results <p>GC–MS demonstrated high glycerol and fatty acid content. ICP-MS demonstrated phosphorus and calcium concentrations of 3000 and 2000&#xa0;ppm respectively. (L), (AEN) and (AENL) increases the microhardness mean values back to basic levels of non-demineralized state when compared to (CH). (L) showed the highest microhardness mean values while (CH) showed the lowest values. After laser irradiation, (L) demonstrated Ca/P ratio identical to that of non-demineralized dentin (1.76).</p> Conclusion <p>Both (L) and (AEN) could restore microhardness of demineralized dentin to normal levels. Combination treatment by the sequence in (AENL) did not demonstrate synergistic dentin strengthening effect. (CH) failed to restore the microhardness of demineralized dentin.</p> Graphical abstract <p>Graphical abstract demonstrating the workflow of dentin samples preparation and testing procedures (created by BioRender)</p> <p></p>