To study the effect of culture medium, organic matter, and salinity on active compounds of lemon balm, a factorial experiment was conducted in completely randomized design with 3 replications. First factor (culture medium at three levels; compost+arable soil, vermicompost+arable soil, and compost+vermicompost+arable soil), second factor (organic matter; humic acid (HA) and effective microorganisms (EM) both 5 per 1000); and third factor (salinity; tap water, 40 and 80mM). Examination of essential oils by the GC method revealed the presence of 34 compounds. These compounds were affected by substrate and salinity. The major constituents of essential oils including Trans-carveol, Carvacrol acetate, Isoborneol, Isopulegol and γ-3-Carene were observed in the combined substrate of arable soil+compost+vermicompost, and the highest levels of 1, 3, 8-P-menthatriene, Citronellol, and γ-Terpinene were observed in the combined substrate of arable soil+compost. With increasing salinity, amount of Trans-carveol, Carvacrol acetate, γ-Terpinene, Isoborneol, Citronellol increased, and γ-3-Carene, Isopulegol and 1, 3, 8-P-menthatriene decreased significantly. The highest percentages of Trans-carveol (21. 85), Isoborneol (12. 90), Carvacrol acetate (11. 78) were observed in the salinity of 80mM in combined substrate of arable soil+compost+ vermicompost. The highest percentage of Citronellol (11. 12) and γ-Terpinene (9. 87) were recorded under compost substrate. In the control and with a combined substrate of arable soil+compost, the highest percentage of compounds, including 1, 3, 8, P-menthatriene (9. 93) was observed. Finally, the combined substrate of arable soil+compost+vermicompost caused the highest percentages of Isopulegol (8. 98) and γ-3-Carene (8. 47). Application of HA could increase the main constituents, in lemon balm.