Melissa officinalis L., popularly known as lemon balm, is an aromatic plant widely used in medicine, cosmetics, and pharmaceutical industries for its essential oil rich in phenylpropanoids, terpenes, and phenolics. This study aimed to assess the effect of growth regulators on the development and physiological and biochemical metabolism of M. officinalis cultured in vitro. Seeds were inoculated in Murashige and Skoog medium and added with the regulators 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) according to six different treatments. After 90 days of culture, plants were evaluated for growth and biochemical and physiological parameters (flavonoids, anthocyanins, and chlorophyll). The balance between regulators interfered with plant growth, which increased in the presence of 0.2 mg L-1 BAP. In this treatment, the plants had greater growth with more leaves, and the biomass production of shoots and roots was higher than the control. Growth regulators did not influence nitrogen assimilation or flavonoid production; however, total chlorophyll and anthocyanin indexes were enhanced by treatment with BAP at concentrations ranging from 0.2 to 0.5 mg L-1. Auxin treatment did not improve root production or growth but favored callus formation when combined with 0.5–3.0 mg L-1 BAP. The results indicate that high BAP concentrations (above 1.0 mg L-1) should not be used in in vitro production of lemon balm.