The purpose of this study was to investigate some possible mechanisms underlying the in vitro antitumor activity of tea tree oil (TTO) on human and mouse breast cancer cells (MCF-7 and 4T1, respectively) and its cytotoxicity on fibroblasts (HFF-1) and on peripheral blood mononuclear cells (PBMCs). TTO High-Resolution Gas Chromatography (HRGC) showed seventeen main constituents, such as Terpinen-4-ol, γ-Terpinene, and α-Terpinene. High TTO concentrations (≥ 600 μg/mL) showed a remarkable antitumor activity, decreasing cell viability and cell proliferation of MCF-7 and 4T1 cells. TTO at 300 μg/mL increased the number of MCF-7 cells in the early stages of apoptosis and increased the BAX/BCL-2 genes ratio. TTO, mainly at 300 μg/mL, decreased cell growth and arrested MCF-7 cells in the S phase of the cell cycle. Lower antitumor concentrations (≤300 μg/mL) evaluated in MCF-7 and 4T1 cells were not cytotoxic to PBMCs and HFF-1. Also, TTO (300 μg/mL) was able to induce cell proliferation in fibroblasts after 72 h, indicating non-cytotoxic effect in these cells. TTO exhibited in vitro antitumor effect on MCF-7 and 4T1 cells by decreasing cell viability and modulating apoptotic pathways and cell cycle arrestment of MCF-7 cells. In this sense, our study provides new perspectives on the potential use of TTO for the development of new alternative therapies to treat topically locally advanced breast cancer (LABC).