This study was performed to determine the chemical composition, antioxidant and cytotoxic effects of essential oils extracted from the aerial parts of fresh (F‐PSEO) and air‐dried (D‐PSEO) Pallenis spinosa. The composition of the oils was analyzed by gas chromatography (GC) and GC/mass spectrometry, the antioxidant activity by free radical scavenging and metal chelating assays, and their cytotoxicity by a flow cytometry analysis. The primary components in both oils were sesquiterpene hydrocarbons and oxygentated sesquiterpenes. F‐PSEO contained 36 different compounds; α‐cadinol (16.48%), germacra‐1(10),5‐diene‐3,4‐diol (14.45%), γ‐cadinene (12.03%), and α‐muurolol (9.89%) were the principal components. D‐PSEO contained 53 molecules; α‐cadinol (19.26%), δ‐cadinene (13.93%), α‐muurolol (12.88%), and germacra‐1(10),5‐diene‐3,4‐diol (8.41%) constituted the highest percentages. Although both oils exhibited a weak radical scavenging and chelating activity, compared to α‐tocopherol and ascorbic acid, D‐PSEO showed a 2‐fold greater antioxidant activity than F‐PSEO. Furthermore, low doses of F‐PSEO were able to inhibit the growth of leukemic (HL‐60, K562, and Jurkat) and solid tumor cells (MCF‐7, HepG2, HT‐1080, and Caco‐2) with an IC₅₀ range of 0.25 – 0.66 μg/ml and 0.50 – 2.35 μg/ml, respectively. F‐PSEO showed a ca. 2 – 3‐fold stronger cytotoxicity against the tested cells than D‐PSEO. The potent growth inhibitory effect of the plant essential oil encourages further studies to characterize the molecular mechanisms of its cytotoxicity.