Olive oil is an important source of mono-unsaturated fat and a prime component of the Mediterranean diet. The beneficial health effects of olive oil are due to both its high content of mono-unsaturated fatty acids and its high content of anti-oxidative substances. The objective of this study was to investigate the basis for the epidemiological information relating to the health benefits associated with the consumption of extra-virgin olive oil (EVOO). For this purpose, the somatic mutation and recombination test (SMART) in wings of Drosophila melanogaster was applied to evaluate the antigenotoxic activity of six different EVOOs from four Spanish varieties (Hojiblanca, Nevadillo, Casta de Cabra, Picual). A two-level approach was followed: (1) determination of the lack of genotoxicity along with the antigenotoxic activity of EVOOs, through antigenotoxicity assays, with hydrogen peroxide as an oxidative genotoxin, and (2) evaluation of the properties of three major distinctive components of EVOOs that could be responsible for their antigenotoxic activity. The EVOOs tested are shown to be non-genotoxic; they exhibited antigenotoxic activity against the effects of hydrogen peroxide. Triolein, tyrosol and squalene did not show genotoxic effects in the proliferative imaginal cells of D. melanogaster. The three components were antigenotoxic when combined with soybean oil, only triolein and tyrosol showed a clear desmutagenic effect when combined with hydrogen peroxide. Our results confirm the safety of EVOOs and their health-protective effects.