The lavender Lavandula multifida L., a medicinal plant grown in arid regions of Tunisia, was recently considered an endangered species; thus, its habitats regressed to some difficult zones in terms of access, such as the watershed of Oued Agareb in central-eastern Tunisia. This species was recorded only in deep and narrow shady Wadi of the watershed and benefited from protection against overgrazing, erosion and sunlight. L. multifida was rarely observed in an open area, such as a plateau or large-bed valley. The plant’s metabolism is linked to its response to environmental conditions, which is of particular interest to understanding the components of the considered population of L. multifida. Consequently, biochemical and antimicrobial analyses have been evaluated. Using gas chromatography-mass spectrometry (GC-MS) analysis reveals that among the 58 compounds identified in L. multifida essential oil extracted from aboveground plant tissues, camphor was the major component (15.68%), followed by 1,8-cineole (14.14%) and alpha-pinene (13.82%). Moreover, it has been observed that Escherichia coli was more susceptible than Staphylococcus aureus to the antimicrobial properties of L. multifida essential oil, while in the case of camphor, S. aureus was more susceptible than E. coli. The protected population of L. multifida exhibits a distinctive vegetative development and growth cycle, resulting in specific secondary metabolites and distinguished antimicrobial activity.