The perennial species , commonly known as St. John's Wort, is well regarded for its medicinal attributes, particularly its strong anti-inflammatory and antidepressant effects. L., commonly known as balsam, is extensively employed in both traditional and contemporary medicine due to its biological properties, although the plant's medicine distribution is limited to Europe and Asia. This study pioneers the investigation of cultivation in a Mediterranean country, specifically Greece, focusing on the effects of irrigation and biostimulants of two distinct genotypes on quantitative (height, drug yield, essential oil yield) and qualitative (essential oil content and composition) characteristics. A field trial was conducted at the experimental farm of the Agrotechnology Department at the University of Thessaly, located in the Larissa region. This study investigated various testing varieties under different irrigation levels and biostimulant applications. The results underscore the importance of customized irrigation and biostimulant strategies in improving yield and quality during the second growing season, establishing a foundation for sustainable agricultural progress. Notably, irrigated treatments significantly increased plant height, dry biomass yield, and essential oil production per hectare. Specifically, the essential oil yields for irrigated treatments were nearly double those of rainfed treatments, with 219 kg/ha for rainfed and 407 kg/ha for irrigated. The genotype played a crucial role in influencing production potential, height, flowering, and essential oil composition, with one variety demonstrating biennial blooming and modified essential oil compounds. While irrigation positively impacted yield, it also reduced certain essential oil compounds while increasing β-pinene content. The effects of biostimulants varied based on their composition, with some enhancing and others diminishing essential oil content. Notably, the biostimulant containing algae with auxin and cytokinin (B2) proved to be the most effective in improving the therapeutic profile. This study offers valuable insights into the cultivation of in a Mediterranean climate, highlighting the necessity for ongoing research into native populations, irrigation levels, biostimulants, fertilization, and other factors that affect crop yield and quality characteristics.