Olive leaf is a byproduct of the olive tree that is rich in phenolic compounds with potential anticarcinogenic effects against various cancers, including breast cancer. Nevertheless, the ingestion or topical application of such plant extracts faces certain limitations. These limitations can be addressed by encapsulating the extracts in nanovesicles to enhance their release and bioavailability. This study aims to develop nanovesicles using Olea europaea leaf extract to exploit its potential anti-cancer properties. Soy lecithin was used to form liposomes for encapsulation of the olive leaf extract. In addition, ethanol and glycerol were added to form ethosomes and glycerosomes, respectively. The antiproliferative effect of both the free extract and the three formed nanovesicles was tested in MCF7 and MCF10A cell lines. To comprehend the mechanisms leading to reduced cell viability after exposure to olive leaf extract and its nanovesicles, levels of reactive oxygen species (ROS), mitochondrial membrane potential, and apoptotic stage were evaluated. The results suggest that both, the nanovesicles and the free extract, are antiproliferative agents against MCF7 tumour cells. However, when examining the impact of olive leaf extract and the formulated nanovesicles on MCF10A cells, no reduction in cell viability was observed. Our findings indicate that the anti-tumour effect of the extract and its nanovesicles may be due to increased oxidative stress, mediated by mitochondrial damage. The mechanism through which olive leaf extract exerts its antiproliferative effect on the breast cancer tumour line implies that apoptosis may be induced by the extract via the involvement of a mitochondria-dependent ROS-mediated pathway.