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Previous studies have reported the effects of pequi oil (Caryocar brasilienseCambess.) in cancer prevention and treatment. However, its biomedical applications are limited due to the hydrophobic nature of the oil. In this study, we used nanotechnology strategies to design and characterize a pequi oil-based nanoemulsion and evaluate its anticancer effects. Our data show that monodisperse (PdI 0.23) pequi oil nanoemulsions (123 nm – hydrodynamic diameter; −15 mV – zeta potential) were prepared successfully using lecithin as surfactant and had physicochemical stability when exposed to stress conditions (pH and serial dilution) or long-term (200 days) storage at different temperatures. Significant dose- and time-dependent antitumoral effects against breast cancer cells (4T1) were also observed (IC50of 299.8 and 173.7 μg/mL after 24 and 48 h respectively, p < 0.001), along with lower cytotoxicity against non-tumoral cells (NIH/3T3) (IC50of 442.7 and 380.3 μg/mL after 24 and 48 h respectively). In addition, we show that in our formulation, pequi oil has a dual role, behaving both as a structural component of the nanoemulsion and as a cytotoxic agent against breast cancer cells. Taken together, our results reveal pequi oil-based nanoemulsion as a promising nanosized platform to be used as an adjuvant tool for breast cancer treatments.