Aflatoxin B1 (AFB1) is a potent mycotoxin that is commonly produced by molds such as Aspergillus (A.) flavus and A. parasiticus. AFB1 is associated with several health adverse effects in humans including mutagenesis and carcinogenesis. Aflatoxin is commonly secreted in the milk leading to deleterious effects on breast tissue and potential nursing infants. However, the effects of aflatoxins, particularly AFB1, on the breast cells are less investigated. In this study, AFB1-associated effects on human breast cancer cell lines (MCF-7) were investigated. AFB1 caused significant cytotoxicity on MCF-7 cells. Such cytotoxicity had a positive correlation with the induction of oxidative stress. In addition, AFB1 caused significant transcriptomic alterations in xenobiotics and drug-metabolizing enzymes, transporters, and antioxidant enzymes. Besides, AFB1 upregulated pro-inflammatory markers such as tumor necrosis factor-α and cyclooxygenase-2 with a significant reduction of mRNA expressions of the immunity-related genes including interleukins 8 and 10.