Olive oil possesses a predominant role in the diet of countries around the Mediterranean basin, whereas it is a known constituent of several sectors of human culture. The polyphenolic composition of olive oil seems to be a key factor in its beneficial biological properties. Based on the above, the aim of this study was to correlate the polyphenolic composition of five extracts derived from a Greek olive oil variety with their antioxidant potency and antimutagenic activities in vitro with chemical-based techniques and cell culture-based assays. According to the results obtained, the polyphenol samples with higher concentration of hydroxytyrosol (HT) were more potent in antioxidant and antimutagenic activity in vitro, as indicated by their ability to scavenge ABTS·+ radical and to protect the strand of plasmid DNA from free radical-induced breaking compared to the corresponding samples with higher levels of tyrosol (T) and its derivatives. However, this observation was not evident in the cell culture model (i.e., the HeLa cervical cancer cell line) to which the tested extracts were administered. Specifically, the T-rich extracts more effectively increased endogenous GSH levels measured by flow cytometry than did the HT-rich compounds. Also, olive oil compounds contributed variously to the expression of genes implicated in the cell antioxidant machinery, as indicated by quantitative PCR. Therefore, the relationship between structure and function in redox regulation is complex and merits the combination of tests. Given that factors like the production and storage regimen of the plants are major determinants of the composition of the generated extracts, we propose that specific conditions should be adopted in order to achieve their maximum biological activity. These results followed by others in the same direction could provide a solid basis for the production of functional foods enriched in olive oil extracts with potential antioxidant action in vivo.