The co-incubation in the culture medium with hydroxytyrosol [3,4-dihydroxyphenyl ethanol (3,4-DHPEA)], the main phenolic compound present in extra-virgin olive oil, and H₂O₂ reduces the oxidative DNA damage in peripheral blood mononuclear cells (PBMC). In this study we investigate, by the comet assay, the ability of 3,4-DHPEA to inhibit the H₂O₂ induced DNA damage when pre-incubated with PBMC and then removed before the exposure of cells to H₂O₂. Low doses of 3,4-DHPEA (10–100 μM) pre-incubated for 30 min with PBMC reduced the DNA damage induced by the treatment with H₂O₂ 200 μM for 5 min at 4 °C. Prolonging the exposure time up to 6 h completely prevented the DNA damage. Furthermore we extensively analysed, by the MTT assay, the anti-proliferative activities of 3,4-DHPEA on breast (MDA and MCF-7), prostate (LNCap and PC3) and colon (SW480 and HCT116) cancer cell lines and correlated these effects with the H₂O₂ accumulation. The concentration of H₂O₂ in the culture medium was measured by the ferrous ion oxidation–xylenol orange method. The proliferation of all the cell lines was inhibited but at different levels: the prostate cancer cells were more resistant to the growth inhibition with respect to breast and colon cancer cells. The ability of the different cell lines to remove H₂O₂ from the culture medium was inversely correlated with their sensitivity to the anti-proliferative effect of 3,4-DHPEA. Therefore, 3,4-DHPEA may act as a chemopreventive agent acting on both initiation and promotion/progression phases of carcinogenesis.