Menthol as an important component of monoterpenes essential oil in peppermint (Mentha piperita L.) is widely applied for medical and industrial uses. In this study, the effect of exogenous applications of chitosan (200 mg/L), gibberellic acid (50 mg/L) and methyl jasmonate (300 μM) was investigated in the main genes of menthol biosynthesis pathways within a 72 h time period using qRT-PCR. Transcript levels of most genes were either unaffected or down-regulated following chitosan treatment relative to control plants. Decreasing of geranyl diphosphate synthase (GDS) and limonene synthase (LS) genes transcript in chitosan treatment could possibly be effective in reducing of limonene level. On the other hand, it seems that an increase in menthone-menthol reductase (MMR) transcription level at 72 h under these treatments had a positive role in increasing the amount of menthol in this plant. Since exogenous application of gibberellic acid (GA3) down-regulated transcript levels of several genes involved in menthol biosynthesis, there is this expectance that GA3 treatment might not have a prominent role in enhancing menthol yield via transcription regulation. Transcript level of the majority genes after methyl jasmonate treatment gradually increased and reached the highest level at 72 h, therefore, it is possible that methyl jasmonate improves medicinal properties of M. piperita.