Peppermint (Mentha × piperita L.) is cultivated for its benefits in pharmaceutical, medicinal, and cosmetic industries. The well-known essential oil of Mentha × piperita L. is widely produced and used all over the world. The aim of present study was to evaluate the impacts of different concentrations of chitosan on the quality and quantity of the essential oil from the aerial parts of peppermint under inoculation of the rhizomes of peppermint seedlings with arbuscular mycorrhizal fungi. Experimental treatments were arranged as factorial design in a completed random block design. The highest essential oil yield (2.4 mL 100 g–1 dry matter) was obtained from the peppermint plants under foliar sprayed at 5 g L–1 chitosan along the inoculum with arbuscular mycorrhizal fungi. For evaluation of phytochemical characteristics, the contents of the main constituents of the peppermint essential oils such as menthol, menthone, etc. (oxygenated monoterpenes and monoterpenes hydrocarbons) under different treatments were analyzed by GC-FID and GC/MS. Results indicated that using chitosan foliar meaningfully raised the amount of menthol, as the major constituent and quality index (>60% v/w), in the essential oil from the peppermint plants inoculation with arbuscular mycorrhizal, however, the plants under the foliar spray of chitosan (without inoculum) revealed the highest amounts of menthone and limonene. In conclusion, we found that the foliar-applied chitosan along inoculation with arbuscular mycorrhizal fungi can be improved quantity and quality active substances of Mentha × piperita L. such as the contents of essential oil, menthol, and balance menthol/menthone.