The goal of this work was to investigate the chemical composition of Aloysia citriodora essential oil and its biological activities as antioxidant, antibacterial, antifungal and anti-inflammatory potentials depending on the collection region. The highest essential oil yields were in Gabes and Kairouan at the Arid bioclimatic zone. Sixty volatile compounds were identified by GC-FID-MS. The major compounds were limonene and spathulenol in Gabes at the Middle Arid. However, (E)-citral (known as geranial) was the major compound of Belli and Kairouan belonging to the Upper Semi-Arid and Upper Arid areas, respectively. The essential oil extracted from the inferior sub-humid bioclimatic zone (Boussalem) had spathulenol and caryophyllene oxide as predominant components. Inversely to DPPH and ABTS, the antioxidant activities using reducing power assay of A. citriodora essential oils significantly increased from the inferior sub-humid (Boussalem) to the Upper Arid (Kairouan) bioclimatic areas. Independently of region, A. citriodora essential oil exhibited an interesting antibacterial, antifungal and anti-inflammatory potential.