Hairy roots are less susceptible to manipulation by changes in medium composition than callus and cell suspension cultures. However, when the individual components of the Gamborg’s B-5 medium were modified one by one, several of them modified the yield of root cultures. Nitrogen (N) source changes, the absence of phosphorus (P), calcium (Ca), and cobalt (Co), and the increase in the sucrose content of the medium increased the level of alkaloids in Datura stramonium hairy root cultures. The use of ammonium as N source provoked the alkaloid excretion into the medium. The addition of different auxins inhibited the alkaloid accumulation, but the addition of abscisic acid (ABA) did not change the alkaloid content of the cultures. The use of MeJa increased the content of hyoscyamine to 100%.