Background: Zamzam water is naturally alkaline and rich in a variety of minerals which may represent a powerful tool for cancer therapy. In this research, the cytotoxic effects of Zamzam water were investigated in human lung cancer (A549) cell line and compared with human skin fibroblasts (HSF). Methods: Two different preparations of Zamzam water were used: Z1, with pH adjusted to 7.2 and Z2, with no pH adjustment. The effects of both treatments on the morphology of the A549 and HSF cell lines were investigated. The cell viability of HSF and A549 cells was identified by the MTT assay and trypan blue exclusion. Detection of apoptotic cells and cell cycle analyses were determined using flow cytometry. Moreover, reactive oxygen species (ROS) were measured for both cell lines. Results: Both Zamzam water treatments, Z1 and Z2 showed reductions in the cell viability of A549 cells. Cell death occurred via necrosis among cells treated with Z2. Cell cycle arrest occurred in the G0/G1 phases for cells treated with Z2. Cellular and mitochondrial ROS productions were not affected by either treatment. Conclusion: Our findings indicate that Zamzam water might have potential therapeutic efficacy for lung cancer.