Chemical composition, antibacterial, antioxidant and cytotoxic activities of (Pistacia khinjuk) hull essential oil (EO) were evaluated in this study. The EO was isolated and analyzed by gas chromatography-mass spectrometry. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined using 6 strains of Gram-positive and negative bacteria. DPPH radical scavenging (DPPH) and β-Caroten Bleaching (BCB) assays were used to measure antioxidant activity of the EO. In vitro cytotoxic activity was measured using MTT assay. Fifty-six compounds representing 99.5% of the total oil composition were identified. In the antibacterial results, Staphylococcus aureus was found to be the most susceptible strain (MIC and MBC = 16 μg/ml). Antioxidant IC50 values were respectively 19.03 ± 0.001 and 49.22 ± 0.005 μg/mL. The IC50 indexes of cytotoxic tests were 29.6, 37.3 and 41.1 μg/mL for MCF-7, PC3 and DU-145 cell lines, respectively. Display omitted •The Chemical composition, antibacterial and cytotoxic activities of P. khinjuk were evaluated.•The minimum inhibitory concentration and the minimum bactericidal concentration were determined using 6 strains of bacteria.•DPPH radical scavenging and β-Caroten Bleaching assays were used to measure antioxidant activity of the EO.•The EO has antibacterial and antioxidant activities and may serve in food and pharmaceutical industries.