An increasing interest is emerging in identifying natural products to overcome drug resistance in cancer patients. In this context, the present study was conducted to investigate the cytotoxic effects of neem plant (Azadirachta indica) oil in three different biological models (breast cancer cell lines, Allium cepa root tip, and mice vital organs). The cytotoxic potential of the neem oil was evaluated with two human cell lines (MCF7 and MDA-MB231) and an Allum cepa root tip bioassay. Histopathological analysis was conducted on the neem oil-treated and untreated control mice. The results revealed an anti-proliferative effect for neem oil on both estrogen receptor-positive (MCF7) and estrogen receptor-negative (MDA-MB231) breast cancer cell lines with an IC50 value of 45.7 and 60 µg/ml, respectively. Also, a significant (p ≤ 0.001) reduction in the average mitotic index and elevated levels of cytogenetic abnormalities were observed in A. cepa root tips treated with different concentrations of neem oil extract compared to untreated roots. Moreover, very low to undetectable histopathological changes in the vital organs (liver and spleen) of mice administered with 15 mg/ml neem oil were also observed. The findings of this study suggest that neem oil extract has the potential to inhibit the growth of different rapidly dividing biological models (breast cancer cell lines and A. cepa root tips) with minimal toxicity to the vital organs of the exposed mice. This could be explored further for the development of novel therapeutics to address the problem of drug resistance associated with different malignancies.