Treatment of St. John’s wort plantlets with 1 µM triacontanol and 2 µM benzyladenine stimulates growth and metabolic processes, being an environmental-friendly approach for optimizing the cultivation of these valuable medicinal plants under controlled conditions. When the two growth regulators (a bioactive cuticular wax constituent and a cytokinin) are applied simultaneously, they act synergistically, enhancing each other’s effect on the biomass accumulation and on certain parameters of the photosynthetic light use efficiency, such as the effective quantum yield of photosystem II and the overall vitality index of the photosynthetic apparatus which performs the conversion of light energy into usable forms for carbon dioxide assimilation. The results concerning the interactions between the two externally applied growth regulators during the early development of St. John’s wort plants may lead to a more efficient cultivation of this herbal medicinal product, including the possibility to modulate the production of pharmacologically active metabolites.