The present study reports the variation in the essential oils (EOs) composition from needles and scale leaves of Pinus halepensis, Cupressus sempervirens and Juniperus phoenicea which collected from three different altitudes at Al-Jabel Al-Akhdar area, Libya. The impact of the altitude on the quantity and quality of EOs was studied. In addition, the antibacterial activity was evaluated using microdilution broth assay technique. The results showed that the highest percentages of the EOs yield were found in P. halepensis with 0.41, 0.60, and 0.43% for the altitudes of 125m (I), 391m (II), and 851m (III), respectively, while the lowest percentages of the yield were found in J. phoenicea, with 0.15, 0.07, and 0.18% at altitudes of  I, II, and III, respectively.  Based on P. halepensis EOs analyses using GC/MS, 35 components have been detected, which represents 89.92, 91.56, and 86.44% for the altitudes of I, II and III, respectively. Furthermore, high percentages from the components of α-pinene, β-pinene, α-terpineol and caryophyllene were identified at the three tested altitudes. For C. sempervirens EOs, 33 components were identified representing 84.94, 93.37 and 99.48% for levels I, II and III, respectively. The highest percentages of the EOs components in this species were α-pinene, terpinen-4ol and α-terpiny acetate at the three tested elevations. However, the detected main components in J. phoenecea EOs were α-pinene, α-myrcene, α-terpinyl acetate and γterpinene. The highest antibacterial activity was observed for the oils from P. halepensisat and J. phoenicea at altitude I against the Agrobacterium tumefaciens, Erwinia carotovora, Corynebacterium fascians and Pseudomonas solanacearum. While the EOs extracted from scale leaves of C. sempervirens obtained from trees growing at level II were the most active against the tested bacteria. The results of this work revealed the impacts of the environmental conditions on the EOs composition which affected significantly in its quantitative and qualitative performances. Consequently, their biological activities were varied considerably. Moreover, the obtained data offer the opportunity to choose EOs with preferential
compound for pharmaceutical, pesticides, perfume and food industries in such important region in Libya.