Due to the rising demand for essential oil in the world market, peppermint has gained an important status among aromatic and medicinal plants. It becomes imperative to optimize its performance in terms of the growth, physiological functioning and biosynthesis of specialized metabolites. A factorial randomized pot experiment was performed using three peppermint cultivars (Kukrail, Pranjal and Tushar) and five levels of leaf-applied nitrogen (N), viz. 0 (control), 0.5, 1.0, 1.5 and 2%. The phenological features, biochemical parameters, viability of root cells, stomatal and trichome behavior were assessed at 100 days after transplanting (DAT). The yield-related parameters, viz., herbage yield, essential oil content, menthol content and yield were studied at 120 DAT. The results revealed that increasing the N doses up to 1.5% enhanced all the studied parameters of peppermint, which thereafter (at the dose above 1.5% N) decreased. The variation pattern of the studied parameters was "low-high-low". Cultivar Kukrail surpassed the two other cultivars Tushar and Pranjal. Among the foliar sprays, the application of 1.5% N increased chlorophyll content and net photosynthetic rate in all three cultivars. Moreover, the essential oil (EO), EO yield and menthol yield of the plant were also increased linearly in all three cultivars as compared with their control plants. Nitrogen application enhanced the trichome size and density of the plants, as revealed through scanning electron microscopy. Furthermore, from the GC-MS studies, the EO content in the studied cultivars increased, particularly in the case of menthol, with the N application. It may be concluded that two sprays of N (1.5%) at appropriate growth stages could be beneficial for improving morphological, physio biochemical and yield attributes of peppermint.