Background: Since alkaloids are nitrogenous compounds, the availability of nitrogen (N) is expected to play an important role in the biosynthesis and accumulation of alkaloids in plants. Nitrogen has been found to increase the content of alkaloids in some of the medicinal plants.

Objective: The aim of this study was to compare the effects of nitrogen bio-fertilizers, conventional nitrogen fertilizer, and nitrogen nano-chelate fertilizer on the content of tropane alkaloids in Hyoscyamus niger L.

Methods: This experiment was conducted in a randomized complete block design (RCBD) with 8 treatments, and 3 replications. The experimental treatments included 2 L.h^-1 nitroxin, 1 kg.h^-1 Azotobacter, and 10 t.h^-1 vermicompost each as bio-fertilizer, 150 kg.h^-1 urea as a conventional chemical fertilizer of nitrogen, and nitrogen nano-chelate fertilizer with three amounts of 3, 5, and 7 percent, and control treatment (without nitrogen fertilizer).

Results: The results showed that the effects of bio-fertilizers, conventional nitrogen fertilizer, and nitrogen nano-chelate fertilizer were significant on the scopolamine and hyoscyamine content in black henbane at the level of 1%. The highest content of hyoscyamine (HYO) was related to the treatments of 5% per hectare nitrogen nano-chelate, and the maximum amount of scopolamine (SCO) was observed in the treatments of 3 and 5% nitrogen nano-chelate fertilizers. On the other hand, the lowest values of hyoscyamine and scopolamine were related to the treatment of 7% nitrogen nano-chelate fertilizer, and nitroxin treatment, respectively.

Conclusion: Generally, the highest yield of alkaloids content in black henbane were obtained by application of 3 and 5% nitrogen nano-chelate fertilizers.