Background and objective Cancer is still a major health problem worldwide, with an estimated 18.1 million new cases in 2018, and it is expected to increase by 75% by 2030. Chemotherapeutic drugs have disadvantages such as toxicity to noncancerous tissues, drug resistance, and recurrence of cancer. Medicinal plants with their active components have great potential as an important source for novel drug discovery owing to their availability, efficiency, and safety. Searching for new strategies to obtain new drugs with higher efficiency and more safety represents an urgent need. Laser light treatment for seeds is known to improve germination, plant growth, and bioactive substance. The goal of this study was to investigate the effect of laser irradiation on improvement of the phytochemicals compounds and biological activities of Balanites aegyptiaca seeds.Materials and methods The effect of laser pretreatment was investigated at different powers, that is, 25, 50, 100, and 200 mW, with two-time intervals for each power (2 and 4 min), on B. aegyptiaca seeds to enhance the germination and antioxidant activity of the methanolic extracts of their dry plant material through different assays and select the most powerful laser pretreatment extract to evaluate the anticarcinogenic activity on different cell lines.Results and conclusion The results bring to light that the most efficient laser treatment for seeds of B. aegyptiaca was at 200 mW/4 min, which induces the highest yield percentage, total phenolic and flavonoid contents, metal chelating, reducing power, as well as free diphenylpicrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activities. Based on these outcomes, the antiproliferative screening assay of the methanolic extracts for the shoots (S) and roots (R) dry plant material of B. aegyptiaca after helium-neon laser treatment at 200 mW for 4 min compared with control was performed on a panel of three cancer cell lines (HepG2, HCT116, and MCF-7) using the sulphorhodamine-B assay, and cytotoxicity was determined using normal BHK fibroblast cell line. Obtained results indicated that these extracts should be regarded as potential anticarcinogenic resources against the HepG2 cell line, displayed moderate activity against MCF-7 and HCT116 cell lines, and exhibited no activity against the growth of the normal BHK cell line. Furthermore, a comparison between these laser-treated extracts, and their mixtures against their control extracts and their mixtures, using the doxorubicin as the reference drug on the HepG2 cell line was in favor of the laser-treated roots and shoots extracts, respectively.