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The aim of this study was to compare different sample preparation methods for the  gas chromatographic determination of the composition of essential oils of Lavandula angustifolia Mill. (Lamiaceae) grown in Jordan. The essential oils obtained by hydro-distillation and supercritical-fluid-extraction (SCFE) from dry flowers and the volatile emitted from the fresh flowers  were determined by solid-phase-micro-extraction (SPME) and  analyzed by GC and GC-MS. In all applied methods, oxygenated monoterpenes dominated the oils. The linalool was the main constituent in the hydrodistilled-, SCF extracted and in SPME detected oils with 35.65%, 33.63% and 22.10%, respectively. The antiproliferative activity of the ethanolic extract, crude lavender oil and of some pure volatile compounds was evaluated using breast cancer cell lines (MCF7, T47D and ZR-75-1). Findings were compared to reference drugs and normal fibroblasts. The ethanolic extract showed potent antiproliferative activity against all breast cancer cell lines under investigation, with minimum toxicity towards the normal fibroblasts. The antiproliferative activity was further estimated for the major constituents against T47D cells and the major activity of the oil was mostly due to linalool and linalyl acetate. This is the first time to investigate the essential oil composition of the aerial parts of L. angustifolia using SPME. The findings of the present study demonstrated qualitative and quantitative variation in the composition of the essential oil among the used methods.