Context: Hyoscyamus niger L. (Solanaceae) roots are rich of tropane alkaloids, such as hyoscyamine and scopolamine are used as the source of raw material for the pharmaceutical industry. Aims: The aim of the present study was to investigate the effect of calcium, magnesium, and iron ions on the production of tropane alkaloids and excretion of riboflavin in H. niger root cultures. Materials and Methods: The calcium, magnesium, or iron enriched/deprived Murashige and Skoog (MS) growth medium were used for culture of H. niger root tissues. The secondary metabolites were quantified using high performance liquid chromatography with ultraviolet detector (HPLC-UV) and fluorimetry techniques. Results: An increased calcium content in the medium unidirectionally reduced hyoscyamine, while increasing scopolamine production with only a moderate impact on riboflavin excretion. Manipulations with magnesium and iron contents in the medium resulted in divergent changes in hyoscyamine, scopolamine, and riboflavin concentrations. Conclusions: Our results show that increased calcium ion content in the Murashige and Skoog medium may be used for the intensification of the scopolamine production in H. niger root cultures.