Abstract Leucojum aestivum L. is a highly demanding pharmaceutical plant which is a natural source of pharmacologically active Amaryllidaceae alkaloids, especially galanthamine and lycorine. To establish an efficient method for the production of these alkaloids Leucojum aestivum plants derived from somatic embryos were subjected to various concentrations of melatonin (N-acetyl-5-methoxy tryptamine), NaCl and melatonin combined with NaCl. The maximum L. aestivum plant biomass was obtained when 10 μM of melatonin was applied to the medium. These plants were characterised by normal growth and did not reveal signs of browning. Enrichment of the medium with NaCl caused a reduction in plant biomass, plant pigment contents (chlorophyll a, chlorophyll b and carotenoids) and an increase in the activities of antioxidant enzymes catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). The addition of melatonin to the medium containing NaCl neutralised the negative effect of salt stress. Moreover, the stimulating effect of melatonin on Amaryllidaceae alkaloids biosynthesis was observed. In plants grown in the medium enriched with melatonin the galanthamine content was, on average, 58.6 times higher, and the lycorine content was 1.5 times higher as compared with the control. In vitro plants obtained from the medium supplemented with melatonin also showed the highest distribution of alkaloids. It is noteworthy that only the plants grown in the medium with melatonin showed the presence of tazettine and chlidanthine, 1,2-dihydro-. This study presents a promising biotechnological approach for the commercial production of alkaloids, including galanthamine and lycorine.