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This study is focused on the analysis of regional variation of the chemical compositions of three Clinopodium menthifolium subsp. ascendens (Jord.) Govaerts Tunisian accession, as well as their inhibition toward fungi and insect pests. The diversity of the chemical constituents and biological activities in front of the aforementioned variations was found to be remarkable. Essential oils were obtained by hydrodistillation of the aerial parts and analyzed by gas chromatography-mass spectrometry (GC-MS). A total of 41, 42, and 30 compounds were identified respectively from Clinopodium menthifolium essential oils harvested from three Tunisian regions, namely Ain-Draham (ADEO), Babouch (BEO), and Tabarka (TEO). All analyzed oils were rich in oxygenated monoterpenes with different major constituents. Piperitenone (34.5%), cis-piperitone oxide (26.1%), and pulegone (47.9%) were the dominant compounds in the three volatile oils, respectively. The antifungal activity was investigated in vitro using six targeted fungal strains (Aspergillus flavus, Aspergillus terreus, Candida albicans, Microsporum canis, Microsporum gypseum, and Trichophyton mentagrophytes). The toxicity and repellency of essential oils were evaluated against the stored product pest Tribolium confusum. The tested samples were differently effective toward the target fungi and the pest depending on the variability of their chemical compositions. BEO exhibited the highest fungitoxic properties toward A. terreus mold, M. canis dermatophyte, and C. albicans yeast (the MIC values ranged from 40 to 400 μg mL -1 ). In addition, the data showed that TEO repelled T. confusum moderately (PR = 42.5% at 2 h after exposure). Concerning the contact treatment, both ADEO and BEO were proved to possess slightly toxic effects toward T. confusum pest (% of mortality 27.5-32.5% at 5% concentration). The results showed that the geographic origin greatly influenced the chemical composition and the associated bioactivities of Clinopodium menthifolium subsp. ascendens.