Many phytochemicals have medicinal properties including anti-inflammatory, antioxidant and cytotoxic activities. The current study aims to estimate and identify the chemical composition of Calamintha fenzlii Vis. leaves volatile oil and to evaluate its cytotoxic, anti-inflammatory and antioxidant properties. The volatile oil (VO) of C. fenzlii leaves was separated using microwave ultrasonic method and the chemical composition was characterized using Gas Chromatography–Mass Spectroscopy method. The anti-inflammatory, antioxidant and cytotoxic activities were determined using a COX inhibitor screening, the DPPH-free radical scavenging and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-cyrboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) cell viability colorimetric assays, respectively. The chemical constituents of C. fenzlii VO were dominated by oxygenated monoterpenoid (96.91%). The major chemical components of C. fenzlii VO were represented by menthone 68.93% and pulegone 23.1%. The cytotoxicity test results revealed that the 23.22, 11.6, 5.8 and 2.9 mg/ml of C. fenzlii VO treatments induced cell death significantly (p ≤ 0.0001) by 70%, while 1.4 and 0.7 mg/ml induced cell cytotoxicity significantly (p ≤ 0.0001) by approximately 50% and 40%, respectively. The VO showed moderate antioxidant activity with an IC50 value of 15.38 ± 0.81 µg/ml. The COX-1 IC50 value was 0.215 µg/ml, while COX-2 IC50 was 0.241 µg/ml. Our data demonstrate for the first time that the VO of the Palestinian C. fenzlii plant possesses cytotoxic, anti-inflammatory and antioxidants effects. It is a good source of therapeutic active compounds that could have potential applications in the nutraceutical and pharmaceutical industries.