Abstract: Objective: The objective of study was to assess the variation of volatiles and biological activities (antioxidant, antimicrobial, cytotoxic and thrombolytic) of essential oils from aerial parts of Mentha longifolia harvested from five regions of Saudi Arabia such as Al-Kharj, Al-Qassim, Dammam, Abha and Al-Madinah. Methodology: Within the regions, the hydro distilled essential oil yield varied from 0.66-1.52% (dry weight basis) with least contribution from Abha and the highest from Al-Qassim populations. The isolated oils were then analyzed by Gas Chromatography-Mass Spectrometry (GC-MS) which confirmed the identification of a total of 18-33 volatile components in different M. longifolia ecotypes, representing 96.7-98.9% of the total oil composition. The oils were analytically characterized by occurrence of carvone (35.30-71.51%) as a principal compound, followed by limonene (5.73-28.45%) and 1, 6 dihydrocarveol (0-12.33%). Results: Results showed that trans-dihydrocarvone, 1,8-cineole, β-caryophyllene, β-bourbonene, germacrene D and bicyclosesquiphellandrene were also detected in considerable amounts. The oils mainly, showed notable (p<0.05) quantitative variations and were dominated by oxygenated monoterpenes (53.76-79.65%) followed by monoterpene hydrocarbons (8.63-32.13%), sesquiterpene hydrocarbons (2.79-18.41%) and then oxygenated sesquiterpenes (0.77-2.18%). The oils (10.0 μg mL–1 concentration) exhibited notable antioxidant potential as assessed by the determination of DPPH• scavenging capacity (54.9-89.7%; IC50 4.4-8.5 μg mL–1) along with total phenols (0.7-4.7% mg GAE/100 g). Moreover, the oils, relative to major compound, carvone and reference compounds, showed moderate antifungal activity against A. niger , A. flavus and F. solani but were weekly active against bacteria such as E. coli and S. aureus. The oils exhibited moderate to good thrombolytic activity with clot lysis of 11.6-68.4% and low cytotoxicity (1.7-5.1%) in vitro. Except, antioxidant assays, the biological attributes of the oils tested were found to be fairly correlated with the contents of oxygenated monoterpenes and/or monoterpene hydrocarbons. Conclusion: Overall, the current findings revealed noteworthy variation in the (bioactives) composition and biological characteristics of the tested essential oils that can be mainly linked to the morphological and biochemical diversity of M. longifolia plants depending on the agro-ecological conditions of the areas/regions selected. The presently examined M. longifolia chemotypes from Saudi Arabia can be explored as a prospective source of carvone-rich essential oil with promising antioxidant, antimicrobial and thrombolytic activities. These results may also provide a scientific basis and support for the ethno medicinal uses of this species in traditional healing.