The current study aimed to extract and identify the chemical compounds of Pistacia lentiscus essential oil (PLEO) from Palestine and to assess its antimicrobial, cytotoxic, and cyclooxygenase (COX) inhibitory activities. The hydro-distilled PLEO was analyzed utilizing the gas chromatography-mass spectroscopy (GC–MS) apparatus. Pistacia lentiscus essential oil (PLEO) was tested for its antimicrobial properties utilizing a broth microdilution method. In addition, an MTS assay was employed to test the cytotoxic activity on a cervical cancer cell line (HeLa). An in-vitro COX inhibition test kit was used to measure the COX inhibitory activity. Twenty-nine molecules were characterized, representing 100% of the total PLEO. Limonene (43.78%), α-pinene (29.45%), and β-pinene (7.54%) were characterized as the major PLEO components. The results revealed that PLEO has broad-spectrum antimicrobial activity against the tested microbial strains and has cytotoxic activity against HeLa cancer cells with an IC50 value of 169.68 ± 2.56 μg/ml. Promising COX-1 and COX-2 inhibitory activities were also demonstrated. The chemical components of PLEO from Palestine were identified here for the first time and showed biological effects against the screened microbes, HeLa cells, and COX enzyme. Further in-vivo investigations are required to assess these activities and other pharmacological effects.