Hyssopus officinalis L. is a medicinal plant containing valuable phytochemicals and is used extensively in the pharmaceutical industry. As this plant naturally contains low levels of these metabolites, plant cell and tissue culture technologies are used to produce them in large volumes. The present study aimed first to evaluate the effects of different types of cytokinins, including benzyl adenine (BA), kinetin (KIN), and thidiazuron (TDZ), at 1 ppm concentration in the culture medium on growth parameters and production of secondary metabolites and photosynthetic pigments in organ culture of H. officinalis; in the second step, the study investigated the effect of different concentrations (0, 0.2, 1, 1.5, and 2 ppm) of the most effective type of cytokinin. The results showed that TDZ was the optimal type of growth regulator compared to BA and KIN as it showed a positive influence on the studied traits; furthermore, its highest concentration of 2 ppm in culture medium was determined to be the best one that enabled the highest production of secondary metabolites and photosynthetic pigments. We conclude that the presence of growth regulators can strongly influence the growth and development of plants in tissue culture conditions.