Mentha Spicata (spearmint), an herbaceous perennial aromatic plant, is the most common herb in tropical and subtropical countries. The essential oil (EO) of Mentha spicata is a valuable source of antioxidants in nutraceuticals and cosmetic industries. In-vitro culturing of Mentha Spicata was done via the micro-propagation technique, and exogenous Mel was used as a plant growth regulator. The field trials involved the plant’s foliar spray of different concentrations of Mel at various time points. The EO of leaves of control and Mel-treated plants (S1-S6) was steam-distilled using a Clavenger-type apparatus. The UV, FTIR, and GC analyses of the EO of control and S1-S6 were recorded. The antioxidant capacity of the EO of control and S1-S6 was measured using a DPPH (1, 1-diphenyl-2-picrylhydrazyl) assay. The nodal explants of Mentha Spicata showed different responses to the shoot proliferation process in the presence of exogenous Mel. The EO yield percentage of control and S1-S6 were in the range of 0.92±0.05% and 0.74±0.03%-0.39±0.04%%, respectively. The UV, FTIR, and GC spectra of the EO of control and S1-S6 presented different numbers of peaks and compounds in the selected range. The EO of control and Mel-treated plants could reduce the free radical DPPH to DPPHH with varying values of IC50. Melatonin could regulate the plant’s growth and development, change the EO yield, improve the EO’s phytochemical profile qualitatively, and enhance the free-radical scavenging capacity of the plant’s EO in a concentration and time-dependent mode. So, it may act as a promising molecule to increase aromatic crop yield with the desired quality.