The olive tree phenolic component oleuropein (OLE) and its derivatives have shown many biological properties, thus representing promising novel therapeutics for the treatment of several diseases, including neoplasia. In this study, we evaluated the activities of OLE and its peracetylated derivative (peracetylated oleuropein, Ac-OLE) against two thyroid tumor cell lines that host genotypic alterations detected in human papillary thyroid cancer. TPC-1 and BCPAP cells were treated with OLE and Ac-OLE, and the effects on viability were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, cell counting, and trypan blue exclusion assays. Antioxidant effects were analyzed by measuring the reactive oxygen species (ROS) in basal conditions and after treatment with hydrogen peroxide (H2O2). Activity of MAP kinase and PI3K–Akt signaling pathways was evaluated by examining the levels of phosphorylated ERK and Akt by western blot. We found that OLE significantly inhibited the proliferation of both cell lines. This effect was paralleled by a reduction of basal phospho-Akt and phospho-ERK levels and H2O2-induced ROS levels. A stronger effect was elicited by Ac-OLE either in inhibiting cell growth or as an antioxidant, in particular on BCPAP cells. Our results demonstrate that OLE and especially Ac-OLE inhibit in vitro thyroid cancer cell proliferation acting on growth-promoting signal pathways, as well as exerting antioxidant effects. Further studies will reveal the potential application as novel targeted therapeutics in thyroid cancer.