Medicinal plants are considered as a very important source of secondary metabolites and therefore bioactive molecules. Our comparative study deals with two species: Hyoscyamus albus and Hyoscyamus muticus harvested in two different climate regions namely: Arris (semi-arid, wilaya of Batna) and Djanet (arid, wilaya of Illizi), respectively. The phytochemical screening revealed that aerial parts of the two species contain almost all classes of secondary metabolites such as flavonoids, quinones, anthraquinones, saponins, tannins, coumarins and alkaloids. The quantitative assessment of alkaloids in hydroalcoholic extracts of the two plants showed that the total alkaloid content varies depending upon the species and climate conditions. The alkaloid level was found to be 0.51% and 0.32% in Hyoscyamus albus and Hyoscyamus muticus respectively. Moreover, the antibacterial test vis-à-vis some pathogen bacteria such as: Escherichia coli, Staphylococcus aureus, Staphylococcus aureus and Pseudomonas aeruginosa showed that both alkaloid crude extracts and hydroalcoholic extracts are moderately active against the tested bacterial strains.