Breast cancer is responsible for one of the top leading causes of cancer deaths among women. Radiotherapy (RT) uses high energy radiation to kill cancer cells, but this method has been reportedly linked to risks of toxicity. Post-therapeutic relapse from RT believed to be caused by its toxicity is one of the challenges encountered during tumour therapy. Therefore, further attention should be devoted to developing novel anti-tumour therapeutic approaches. The role of low-level laser therapy (LLLT) in breast cancer management is to alleviate the side effects arising from RT, instead of acting against the tumour cells directly. This study investigated the effects of low-level laser (532 nm), as well as single and fractionated irradiation, on breast cancer MCF 7 cell line. Additionally, this study assessed the most effective laser parameter for fractionated irradiation. The MCF 7 cells were irradiated with green laser power at 1.5, 45.0, and 100.0 mW with a spot size diameter of 0.7 mm for 1, 5, 10, and 15 min. The irradiation was carried out in single, double, and triple fractionation separated by 5- and 10-min intervals in between the fractional regimes. The laser output of 100 mW showed a promising potential in killing cells with single fractionation. However, as the irradiation was fractionated into two, power of 1.5 mW appeared to be more effective in cell death, which contributed to the lowest percentage cells viable of 31.4% recorded in the study. It was proven that fractionated regime was more successful in tumour cell death.