Breast cancer remains the most common malignancy among women worldwide, contributing to high morbidity and mortality rates. Many anti-cancer drugs have been derived from medicinal plants, and frankincense from is notable for its anti-inflammatory, anti-neoplastic, and anti-carcinogenic properties. Using gas chromatography/mass spectrometry (GC/MS), 48 components were identified in essential oil, and the major constituent was α-pinene (35.81%). In this study, we investigated the anti-tumor effects of frankincense essential oil (FEO) and its nano-formulation with chitosan (FEO-CSNPs) using in vitro breast cancer models (MCF-7, MDA-MB-231, and 4T1 cells) and in vivo mouse mammary carcinoma (4T1) models (Balb/c). The results showed significant reductions in cell viability. At 10 μg/mL, the FEO showed the highest reduction in the C-166 cells, while at 100 μg/mL, the FEO exhibited a stronger cytotoxicity in the MDA-MB-231 and 4T1 cells compared to the FEO-CSNPs and CSNPs. The FEO-CSNPs exhibited cell growth arrest in the S, G2/M, and G1/S phases in the MCF-7, MDA-MB-231, and 4T1 cell lines (36.91%, 23.12%, and 33.58%), in addition to increased apoptosis rates in the MCF-7, MDA-MB-231, and 4T1 cell lines (33.04%, 36.39%, and 42.19%). The wound healing assays revealed a decreased migratory ability in the treated cells. The in vivo experiments in the balb/c mice demonstrated a reduction in the tumor volume, with a histopathological analysis confirming extensive tumor necrosis. Moreover, the FEO and FEO-CSNPs showed notable antioxidant and arginase activity. The gene expression analysis via qPCR indicated the upregulation of tumor suppressor genes and the downregulation of oncogenes. These findings suggest that FEO and its nano-formulation, particularly in the form of FEO-CSNPs as an oral formulation, display enhanced efficacy, warranting further preclinical and clinical research to develop innovative treatment strategies.