Mentha × Piperita L. or peppermint belonging to the family Lamiaceae (mint) is an important herb and useful in pharmaceutical, medicinal and cosmetic industries. In this study, the interactive effects of the foliar application of chitosan and inoculation of the rhizomes of peppermint seedlings with arbuscular mycorrhizal fungi (AMF) on growth and of the contents of menthol and balance of menthol/menthone of the essential oil from M × Piperita in a randomized complete block design (RCBD) were evaluated. The essential oil from the aerial parts (stem and leaves) of this herb was analyzed by GC-FID and GC/MS. Results indicated that the experimental treatments especially the inoculation of the peppermint plants with AMF influenced fresh and dry herbal weights and dry matter of the herb. The major constituents of the essential oil of M × Piperita were menthol and menthone belonging to oxygenated monoterpenes. The highest contents of essential oil yield, menthol and balance of menthol/menthone of the essential oil from M × Piperita were obtained from the peppermint plants under foliar spraying of chitosan and inoculation with AMF, however, the herbs under the foliar application of chitosan (both concentrations) without inoculation showed the highest percentages of menthone and limonene. The results indicated that elicitation of the peppermint with chitosan and inoculation of the peppermint plants with AMF has positive effects on dry herbal weight and secondary metabolites and can be improved the quantity and quality active substances of M × Piperita L.