The non-directed approach to drug discovery has been used for many years in the field of pharmaceuticals. It is based on the investigation of the pharmacological effects of chemical substances, including secondary plant metabolites. The purpose of this work was to investigate the antiproliferative effect of the essential oil from the plant of the genus Lavandula angustifolia, which is cultivated in Greece. Chemical analysis of the essential oil showed enrichment with flavonoids and phenolic compounds. The growth inhibitory effect of the essential oil was examined on colon cancer cell lines. The SRB method was used to assess the effect of the oil on cell viability. The results showed that the essential oil inhibits cell growth in colon cancer cell lines of murine (CT26) and human (HT29) origin. Cell cycle analysis and the ability of the oil to induce apoptotic cell death was assessed by flow cytometry. Following a 24-hour exposure to an oil concentration of 0.035% v/v approximately 30% of CT26 cells were undergoing apoptosis. These findings were also confirmed by fluorescence microscopy, after staining and observing the characteristic lesions of the nucleus of apoptotic cells. This observation was more pronounced in CT26 cells, as in HT29 the percentage of apoptotic cells was significantly lower. Further studies are necessary, in order to both understand the underlying mechanisms and to uncover additional bioactivities of this essential oil. The research project was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “1st Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment” (Project Number: HFRI-FM17C3-2007).